Genetic interactions among components of the messenger RNA biogenesis machinery in the yeast Saccharomyces cerevisiae
Ian Matthew Donaldson
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular and Medical Genetics University of Toronto
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GENETIC INTERACTIONS AMONG COMPONENTS OF THE MESSENGER RNA BIOGENESIS MACHINERY IN THE YEAST SACCHAROMYCES CEREVISIAE Ian Matthew Donaldson, Degree of Doctor of Philosophy, 1999, Department of Molecular and Medical Genetics, University of Toronto
The overall goal of this thesis was to characterize mutations in the zinc-binding domain (ZBD) of the iargest subunit (Rpo2lp) of yeast RNA polymerase iI and then use these mutations to find new genetic interactions with other components of the messenger RNA biogenesis machinery. The first data chapter demonstrates that the zinc stoichiometry of RNA polymerase II (RNAP KI) is 7; consistent with the number of zinc-binding motifs present in the enzyme. Mutations in the Rpo2Ip-ZBD reduced RNAP II activity without visibly altering association of subunits required for this activity.
The second data chapter describes the use of an RpoSlp-ZBD mutant in a synthetic- lethal screen designed to identify other components of the transcriptional machinery. Two mutant genes were identified; an allele of SRBS (a known component of the RNAP II holoenzyme) and an allele of GCR3 ( a factor that had previously been implicated in the expression of glycolytic genes). No evidence was found to suggest that Gcr3p is a component of the holoenzyme; its effect on in vitro transcription appeared to be marginal and possibly indirect. Gcr3p was identified (by another group) as the largest subunit of the nuclear cap-binding complex and was shown to play a role in efficient splicing. The allele of GCR3 identified in this synthetic-lethal screen also had an effect on in vitro spiicing. 1 hypothesize that this defect, coupled with the transcriptional defect conferred by the zinc- binding domain mutation is sufficient to explain the synthetic-lethality observed between the two. The third data ctiapicr de.scribes an attempt to determine if SrbSp also ploys a role in
~p!i~-lng€,nrncts of thc mutant SRB5 strain were deficient in splicing activity. This effect u ltlicly an indirect one. possibly mediated by the effect of the SM5 mutant allele on the pnc cspresion of one or more splicing factors. Extracts made from Rpo2lp-ZBD riiutrints also wcre rcduced in splicing activity raising the possibility that splicing activity is w ns i t lvc to de fects in components of the transcriptional machinery . ACAWOWLEDGEMENTS
I would l&e to thanlc my supervisor Jim Friesen for his many years of tireless suppoa encouragement and enthusiasm. I would also Like to acknowledge the past and pmcnt members of my cornmittee for their guidance; Tom Yager. Ben Blencowe, Jacqueline Segall and Vincent Giguem.
1 am gratefd to Jacques Archambault, Emmanuel Maicas. Keith Schappert. Shahrzad Nouraini, Mike Drebot, Dave Jansma, Jiusheng Wu, Mark Toone, Sara Petersen-Bjorn, Brian Bobecko, Scott Houliston. Debbie Field, Vicki Lay, Deming Xu, Tina Harrington. Alia Ahmed, Dorian Anglin. Yan Xu, Andreas Zurlinden. Manuela Moser, Siro Trevisanato and Driss Talibi. These were the many members of the Friesen lab, past and present, that 1 got to how. They made the Friesen lab an intereshg and stimulating place to work in. My especial thanks go to Debbie and Shahnad for their encouragement. 1 am also indebted to Chris Koth, Saily Hemming, Steve Orlicky, Don Awry, Johnson Wong and Mike Kobor for al1 their helpful guidance.
There are also the indispensable friends who provided a haven; Monis Manoison, Petra Kuehl, Mariano Radaeli, Pascale Rousseau and Keith Neil. 1would especially like to thank Moms for his advice and fkiendship.
Lastly, and most importantly 1 would like to achowledge my family. Tney have ail been waiting patiently a long thefor this. Most of aii, 1 wish to thank my wife Katerina who has walked bai& me the whole way. TABLE OF CONTENTS
Abstract ...... ri Acknowiedgements ...... iv Table of Contents...... v List of Tables...... vii List of Figures...... viii List of Abbreviations ...... x A note on RNA polymerase nomenclature...... ai
CHAPTER 1 A review of the events of mRNA biogenesis and their coordination Overview and Scope...... Transcnptlon initiation- ...... Transition from the pre-initiation complex to the elongation complex ...... Initiation of transcription and the biogenesis of mRNA...... S'-end capping of the pre-mRNA ...... Capping and mRNA biogenesis ...... Splicing...... Splicing and mRNA biogenesis ...... Processing of the pre-mRNA 3'-end...... 3'-processing and the biogenesis of mRNA ...... Temination of transcription and mRNA biogenesis ...... RNA export from the nucleus...... Summary of mRNA biogenesis ...... Thesis summary...... References......
CEIAPTER II Zinc stoichiometry of yeast RNA polymerase II and characterinition of mutations in the zinc-binding domain of the largest subunit Abstract ...... II-2 Introduction ...... II-3 Matenals and Methods ...... 11-8 Results ...... 11-18 Discussion ...... IL38 References...... 11-46
CHAPTER m A synthetic-lethal screen for factors that interact huictionally with RPO2l Abstract ...... III-2 Introduction...... III-3 Materials and Methods ...... ïïl-6 Results ...... ,...... ïïI-23 Discussion ...... ,...... JII-56 References...... ,, ...... III-64
CHAPTERIV Characterizaiion of a mutant srb5 spking defec t and the effect of an RNAP II carboxyl-terminal domain peptide on yeast in vitro splicing . Abstract ...... Introduction ...... Materials and Methods ...... Results ...... , ...... Discussion ...... References...... ,......
CHAPTER V Thesis Summary and Future Dktions Zinc-binding domain mutants of RPOZI ...... ,...... V-2 Pointers to a successful synthetic-lethal screen ...... V-3 GCR3 and RNAP II...... V-5 GCR3...... V-6 SRBS ...... V-9 The RNAP II CTD and splicing ...... V-10 References...... V-13 LIST OF TABLES
CHAFTER 1 Table 1.1 Components of the yeast basal transcription machinery...... I-9 Table 1.2 Cornponents of the yeast RNAP II holoenzyme ...... 1-12 Table 1.3 Capping enzymes and the cap-binding complex of yeast...... 1-27 Table 1.4 Components of the 3'-processing machinery in yeast ...... 1-43
CHAPTER n Table 2.1 Yeast strains used in this study ...... II-19 Table 2.2 Plasmids used in this study..... ,...... II- 11 Table 2.3 Zinc stoichiornetry of RNAP II ...... II-21 Table 2.4 Summary of quantification of Figure 2.6 ...... II-32 Table 2.5 RNAP II zinc-binding capacity ...... II-39
CHAPTER m Table 3.1 Yeast strains used in this study...... ID-7 Table 3.2 Plasmids used in this study...... III-9 Table 3.3 Synthetic-lethal screen results ...... III-27 Table 3.4 Summary of synthetic-lethal rescue results...... III-30
CHAPTER IV Table 4.1 Yeast strains used in this study...... IV4 Table 4.2 mRNA expression levels of some splicing genes are decreased in a Am65 mutant with respect to a WT strain ...... IV-30
vii LIST OF FIGURES
CHAPTER 1 Figure 1.1 An overview of mRNA biogenesis ...... 1-5 Figwe 1.2 Initiation of aansrription and transition to the elongation a>m plex. 1-8 Figure 1.3 The steps involved in capping the S'end of nascent pre-mRNA ... 1-24 Figure 1.4 A summaq of the components involved in yeast pre-mRNA 3'-processing ...... 1-42
C-R II Figure 2.1 ZBD mutant phenotypes ...... , ... -....- - --. .. - ...... -- ...... -...11-7 Figure 2.2 RNA polymerase II preparation used for atornic-absorption spectroscopy ...... 11-20 Figure 2.3 Zinc-blot andysis of the zinc-bhding domain (ZBD) of Rpo2lp in its wild-type and mutant foms...... II-24 Figure 2.4 Effect of a C 110s substitution mutation on Rpo2 lp steady-state levels as detennined by protein-blot analysis...... -...... II-27 Figure 2.5 Transcription activity of ZBD mutant extracts...... II-30 Figure 2.6 Analysis of stoichiometry of RNAP II immunoprecipitated from wild-type and mutant ce11 extracts ...... 11-33 Figure 2.7 Cornparison of subunit profile and activity in WT and mutant Ml RNAP II preparations ...... II-36
CHAPTER III Figure 3.1 WT RP02I plasmids ...... III-14 Figure 3.2 Synthetic-lethal screen method ...... III-25 Figure 3.3 Rpo2 1p steady-state levels in srb5-100 and gcr3-lm mutant strains...... III-33 Figure 3.4 Identification of the gcr3-100 mutation ...... III-38 Figure 3.5 Extracts of gcr3-100 have an in vitm promoter-directed . . transcrrption defect ...... III4 Figure 3.6 Extracts of gcr3-I tXl have non-specinc RNA polymerase II activity equivalent to that of WT exuacu...... DI43 Figure 3.7 Extracts of 4cr3have WT Ievels of promoter-directed transcription. . activity ...... ,...... Dl47
viü Figure 3.8 HA-tagged Gcr3p does not CO-immuwprecipitatewith RNA polymerase II ...... El-49 Figure 3.9 HA-tagged Gcr3p does not interact with the CII) of the largest subunit of RNAP II ...... ILI-51 Figure 3.10 Extracts of gcr3-100 have reduced in vitro spïicing activity...... III-55
CHAPTERIV Figure 4.1 The s tbS-100 mutation ...... N-12 Figure 4.2 Extracts of srb5-la0 are deficient in promoter-spedic transcription . . activity ...... IV-14 Figure 4.3 Extracts of srb5-100 are deficient in splicing activity...... IV- 17 Figure 4.4 Recombinant Srb2p and SrMp do not restore the splicing activity of srb5- 100 extracts...... N-19 Figure 4.5 A CfD peptide inhibits spticing activity of HeLa extracts...... N-23 Figure 4.6 Addition of CïD peptide to yeast whole-cell extract has no effect on . . . . in vitro splicing activity ...... N-25 Figure 4.7 Extracts of RP021-ZBD mutants have reduced splicing activity .... IV-27 LIST OF ABBREVIATIONS
3' 3 prime 5' S prime 5-FOA 5-fluoroorotic acid aka also known as bp base pair CBC cap-binding complex CBP cap-binding protein CF 3'-end cleavage factor of pre-mRNA
CP* counts per minute CPSF cleavage-polyadenylation stimulatory factor CTD carboxyl-terminal domain (of the RNAP II largest subunit) DNA deoxyribonucleic acid DSE down-stream element GST glutathione-S-tram ferase GTF general transcription factor hnRNP heterogeneous nuclear RNA-protein particle hrs. hours kb kilobase pairs kDa kilocialtons LSM low-sulfate medium MED medator min. minutes mRNA messenger RNA mRNP messenger RNA-protein particle NPC nuclear pore complex NSM no-sulfate medium nt nucleotide ORF open reading kame PAP P~Y(A) polyme- PIC pre-initiation complex PF pre-mRNA polyadenylation factor rlWA ribosomal RNA RNA ribonucleic acid RNAP II RNA polymerase II LIST OF ABBREVIATIONS (continued)
RNAP m4n RNA polymerase lacking subunits RpMp and Rpb7p RPW Rpb 1 protein: largest subunit of yeast RNAP II (same as Rpo2 lp) RPBI gene encoding the largest subunit of yeast RNAP II (same as RP021) Rpo2 1p Rpo2 1 protein: largest subunit of yeast RNAP II (same as Rpb Ip) RP02I gene encoding the largest subunit of yeast RNAP II (same as RPBI ) RRM RNA recognition motif SC synthetic complete snRNA small nuclear RNA SR serine-arginine SRB suppressor of RNA polymerase B TAF TBP associated factor TBP TATA-box binding protein tRN A transfer RNA ts temperature sensitive U snRNA U-nch srnall nuclear RNA wr w ild-type YPD yeast peptone dextrose medium ZBD zinc- binding domain A note on RNA polymerase nomenclature
Three nomenclatures have ken developed to describe the subunits of RNA polymerase from yeast. Al1 of these nomenclatures (especially the first two) appear throughout the thesis for two reasons; 1) each nomenclature appears in the Iiterature which 1 cite and 2) mutant alleles have ken individually assigned to each nomenclature (thus, even though the genes RPBI and RPO2l are equivalent, the mutant alleles rpbl-l and rpo2l-1 are not equivalent).
1) RPO Nomenclature (Ingles et al., 1984) Genes encoding the RNA polyrnerase 1, II and III subunits are narned RPOIx, RPOZx and RP03x respectively where "x" indicates the relative size of the subunit. Therefore -21 and RP022 exode the first largest and second largest subunits of RNA polymerase II. The corresponding proteins are Rpo2lp and Rpo22p.
2) A/B/C Nomenclature (Young and Davis, 1983) Genes encoding the RNA polymerase 1, II and III subunits are named RPAx, RPBx and RPCx respectively where "x" indicates the relative size of the subunit. Therefore RPBI and RPB2 encode the first largest and second largest subunits of RNA polymerase II. The corresponding proteins are Rpb 1 p and Rpb2p.
3) Molecular weight nomenclature (Riva et al., 1986) Genes encoding the RNA polymerase 1, II and iII subunits are narned RPAx, RPBx and RPCx respectively where "x" indicates the molecular weight (kDa) of the subunit. Therefore RPC34 and RPC3 I encode the seventh largest and eighth largest subunits of RNA polymerase III. The corresponding proteins are Rpc34p and Rpc3lp. This nomenclature is rarely used to describe RNAP II subunits and is usually reserved for RNAP III.
In addition, subunits which are common to al1 three RNA polymerases are named according to the polymerase in which the subunit was first found. Therefore, the sixth Iargest subunit of RNAP Ii is called RP026 or RPB6 even though it is also found in RNAP I as the eighth largest and in RNAP III as the eleventh largest subunit. This last point is rarely of relevance to this thesis since 1 do not discuss the shared subunits in any depth.
xii Chapter 1 A review of the events of mRNA biogenesis and their coordination Overview and scope Pre-messenger RNAs (pre-mRNAs) in eukaryotic cells are transcribed from a DNA template by RNA polymerase II. These pre-messenger RNAs undergo processing to become mature mRNAs before they can be efficiently transported from the nucleus to the cytoplasm and translated by ribosomes into protein. These two steps of mRNA biogenesis (transcription and processing) are canied out by several protein complexes which are likely to be inter-related temporally and spatiaiiy. The transcription of pre-mRNAs can be divided into three stages. First, an initiating RNA polymerase II (RNAP II) complex binds to the DNA at a promoter element and initiates transcription. Second, an elongating RNAP II complex moves dong the DNA by processively adding nucleotides to the 3'-end of the growing pre-mRNA using the DNA antisense strand as a template. Third, signals encoded by the DNA cause RNAP II to terminate transcription and dissociate from the DNA. Proteins associated with RNA polymerase that modify its activity are different for each of these three stages. While the proteins that make up the initiation complex have been studied thoroughly, the contents of the elongation and termination complexes and how they are related to the initiation complex is not well understood. The process of convening pre-mRNAs to mature mRNA involves three types of modification. First, a methylated guanosine cap structure is added to the S'-end of al1 eukaryotic pre-mRNAs. Second, pre-mRNAs are cleaved at a point upstrearn of their 3'- terminal ends and a poly-adenosine tail is added by polyadenosine polymerase to the new 3'-end of the pre-mRNA. Third, those pre-mRNAs that contain non-coding, intervening sequences (introns) are "spliced" such that introns are removed and coding sequences (exons) are joined together as one contiguous unit. The majority of these modifications to pre-mRNA likely occur CO-transcriptionaily in vivo, even though each of them can be uncoupled from transcription in vitro (Le.; a pre-mRNA made in vitro by a bacteriophage polymerase can be capped, spliced, cleaved and polyadenylated by eukaryotic factors). Recently, severai Lines of evidence have converged to support the idea that transcription
and pre-mRNA processing are coupled to one another in vivo. These events might be closely related both temporally and spatially in a mRNA "factory-like" complex. The coordination of events seems to center on two major structures: the C-terminal dornain (CTD) of the largest subunit of RNA polymerase II and the cap-binding complex that recognizes the 5'-end cap structure, which is unique to RNA polymerase lI transcripts. In the following sections, 1 wiil review the proteins and complexes that are involved in each step of mRNA biogenesis in the yeast Saccharomyces cerevisiae (the mode1 system primarily used in the thesis research). Foiiowing the description of each step, 1 will review how that step is coordinated with the other steps with an emphasis on the CTD and the 5'- cap-binding complex. 1 will describe differences between the yeast and mammalian systems where appropriate. Figure 1 provides a schematic of mRNA biogenesis and serves as a synopsis of this introduction. Figure 1.1: An overview of mRNA biogenesis. Numbers beside each of the processes represent the relevant section of the introduction in which they are described. Ovals represent the RNA polymerase II holoenzyme; the differences in shading at different stages represents changes in holoenzyme contents from one stage to another. The CTD of RNAP II is represented in its phosphorylated form by a circled P. The DNA template is drawn as a single line bound by double-slanted lines. Exons are indicated by black and white boxes on the DNA or RNA. The transcription start site and 3'-processing sites are indicated by arrows that are parailel and perpendicular to the template, respectively. The
S'-cap structure of RNA is represented by a (0) bound by the cap-binding comptex (square box). The spliceosome is represented by a rounded rectangle and the 3'-processing machinery by a gray circle. Mature mRNA forms MAparticies (mm)that are exported to the cytoplasm through the nuclear pore complex (NPC). The phosphorylated CTD and the 5'-cap-binding complex are involved in coordinating the events of mRNA biogenesis as indicated by the arrows. Numbers beside each of these arrows indicates the section of the Introduction where that particular connection is discussed. In addition, a summary of the evidence implicating each of these structures in mRNA biogenesis is tisted below . The CTD and coordination of mRNA biogenesis. 1) The enzymes responsible for capping the S'-end of nascent pre-rnRNA are associated with the phosphorylated form of the C-terminal domain of the largest subunit of RNA polymerase in both mammalian and yeast systems (Cho et al., 1998; Cho et ai., 1997; McCracken et al., 1997). 2) A nurnber of splicing-related factors are associated with the mammalian CID (Du and Warren, 1997; Kim et al., 1997; Mortillaro et al., 1996; Yuryev et ai., 1996) including SRcypKASP10 and CASPI 1 (Bourquin et al., 1997; Tanner et al., 1997), SAF- B (Nayler et al., 1998), SCAFS (Patturajan et al., 1998), PSF and p54nrb (Ernili, 1997). Furthemore, either the CTD peptide or antibody directed against it are able to inhibit splicing in vitro (Chabot et al., 1995; Vincent et al., 1996; Yuryev et al., 1996). The CTD is also able to inhibit splicing when expressed in vivo (Du and Warren, 1997). It is unclear if the CTD is similarly rdated to splicing in yeast. 3) The marnmaiïan cleavage and polyadenylation factor (CPSF) and mammalian cleavage stimulatory factor F (CstF) that are involved in 3'-end formation of the mRNA are associated with the CTD (Dantonel et al., 1997; McCracken et al., 1997). It is unclear if a similar relationship exists in yeast. The cap-binding complex and coordination of niRNA biogenesis. 1) The cap-binding complex that recognizes the newly capped pre-mRNA is required for efficient splicing of introns in both the yeast and mammalian systems (Izaurralde et al., 1994; Lewis et al., 1996b; Lewis et al., 1996a). 2) The cap and cap-binding complex are required for efficient Scleavage in the marnmalian system (Flaherty et al., 1997). It is not clear at this point if this relationship exists in yeast. O Initiation 1.1- : .
Figure Key Splicing... . -al:.' 0 initiating RNAP Il complex elongating RNAP II complex terminating RNAP II complex
@ phosphorylated CTD 5-end cap-structure of mRNA 0 cap-binding complex
O splicing proteins 3'-end cleavage and polyaderiylation proteins Temimation o. 4
polyadenylation *4.1. hnRNPs, ?
formation of mRNP 1.1 Transcription initiation
RNAP II
The synthesis of RNAs that encode proteins and some small nuclear RNAs is catalyzed by RNA polymerase II (RNAP II) in eukaryotes. Yeast RNAP II is composed of 12 individual subunits and aU the genes encoding these subunits have been cloned (see Table 1.1 and (Myer and Young, 1998)). There is a striking degree of conservation between each of these subunits and their human orthologues, which fonn a similar 12 subunit enzyme. One of the first features recognized as king highly conserved by al1 eukaryotes was the C-terminal domain (CTD) repeat structure of the largest subunit (Rpblp or Rpo2 lp) (Allison et al., 1985; Corden et al., 1985). The CTD consists of a heptamer sequence (YSPTSPS) that is repeated 26 times in yeast and 52 times in humans. The mD is unique to RNAP II; neither RNAP I nor RNAP iTi have an equivalent of the CTD repeat domain. Purified yeast RNAP II is able to synthesize RNA in vitro from nucleotides using DNA as a template, however, by itself it lacks the ability to bind and initiate transcription from specific promoters. This activity requires the presence of the generai transcription factors (GTFs).
GTFs The generd transcription factors that are required to direct in vitro promoter-specific initiation of transcription by RNAP II include transcription factor IID (Tm),TFIIB, TFIIF, TFUE and TFIIH (see Figure 1.2; reviewed in (Roeder, 1996)). One additional factor, Tmis required only for efficient transcription in in vitro systems that are not completely composed of well-defined, reconstituted transcription factors. Each of these factors was onginally isolated as a biochemical fraction that could be added in a step-wise fashion, dong with RNA polymerase, to a DNA template to form a pre-initiation Figure 1.2: Initiation of transcription and transition to the elongation complex. Transcription is initiated hom a DNA promoter at nucleotide + 1 (by definition). The TATA promoter element nucleates formation of the pre-initiation complex at the promoter by binding the TATA-binding protein (TBP) . Other general transcription factors (TFIIA, B , F, E and H) and RNA polymerase ïi either assemble at the promoter sequentially or are recmited together with TBP as a holoenzyme (see text). Components of the mediator are probably recruited as part of the holoenzyme but are required only for mediating the effects of transcriptional activators and suppressors. Subsequent steps lead to melting of the DNA at the promoter, initiation of transcription and promoter clearance by the RNA polymerase elongating complex. During this transition to elongation, some general transcription factors (GTFs) may remain associated with the promoter (TFIIA, D) to initiate subsequent rounds of transcription. Other GTFs (B, E and H) dissociate from the elongating polymerase. At least one GTF (TFIIF) is associated with the elongating polymerase dong with other elongation factors such as TFIIS. The exact contents of the elongation complex is not known but may include proteins that were originally associated with the mediator (indicated by a circled '?'). Protein-components of the pre-initiation complex are listed in Tables 1 and 2. The pre- initiation complex shown here is not necessarily to scale nor representative of protein- protein contacts made between factors. For a more detailed review of these aspects see (Orphanides et al., 1996; Robert et al., 1998). Hokenryme bi-s .. or assembles seqhially
Mediator 1
closed pre-initiation complex (PIC)
v open PIC 'NWs required for lnitiat ion CM.phgsphorylation- .?
Elongation
Residual promoter Elongation complex complex - Table 1.1: Com~onentsof the veast basal transcrivtion machinerv ma art 1 of 2)
Gene Size Essen tial Function Features Interactions Structural References
Core RNAP II RPBI/RPO21 basal traiiscription conserved CTD RPB2 basal transcription RPB3 basal transcription RPB4 promoter-directed t rs Rpb7p RPBS basal transcription also a subunit of pol 1, III R PB6/R PO26 basal transcription also a subunit of pol 1, III RPB7 basal transcription R~b4~ RPB8 basal transcription also a subunit of pol1,111 RP09 accurate initiation RPBlO basal transcription also a subunit of poli, III RPBll basal transcription WB12 basal transcription
TFliD (yeast biochemical fraction d) TBPlISPT15 27 Y binds TATA element also required by pol 1, III Sua7p, TAFs Gal4p TAFs TBPl associated factors are not listed here
TFIIB §(yeast biochemical fraction e) SUA7 38 Y start-site sclcction hTFl10 orthalog (35%) Tbplp, Rpblp, ZBD (19) Gal4p, Ssul?
TFIlF §+(yeast biochemical fraction g) SSU7l/rFCI 82 Y elongation factor hRAIJ74 ortholog (27%) TFC2 47 Y elongation factor hRAP30 ortholog (31%) TFG3IANCl 27 N same as yTAF30 also component of SWI/SNF Table 1.1: Comvonents of the veast basal transcxivtion machinerv (vart 2 of 2)
Gene Size Essential Function Features Interactions Structural References name kDa (Y/N) motifs
TFIIE (yeast biochemical fraction a) TFA l 55 Y promoter clearance TFIIH, Calllp ZBD TFA 2 37 Y promoter clearance TFIIH, Galllp
TFllH (yeast biochemical fraction b) involved in promoter clearance and nucleotide excision repair (NER) TF01 73 Y NER hBTF2 ortholog (26%) TFB2 59 Y NER TFB3 32 Y NER Kin28p RING motif TFB4 37 Y RAD3 90 Y DNA helicase SSLl 52 Y NER SSL2/RAD25 95 Y DNA helicase TFIIK §(a subcomplex of TFIIH) KIN28 35 Y cyclin-dependent ortholog of MO15 CTD kinase CCLl 45 Y Kin28 cyclin partner; ortholog of cyclin H
TFllA TOA 1 32 Y TOA2 13 Y
Notes and references to Table 1.1
This Table was adapted from (29). Abbreviations used: ZBD, zinc-binding domain; CTD,C-terminal domain of RPBl; trs, transcription; NER, nucleotide excision repair * indicates that this component wns identified as part of the mediator (see reference 3 in Table 1.2) 9 indicates that this component was identified as part of the original holoenzyme (see reference 1 in Table 1.2), Notes and references to Table 1.1 (continued)
Young, R. A., and Davis, R. W. (1983) Scierice 222(4625), 778-82 Ingles, C. J., Himmelfarb, H.J., Shales, M., Greenleaf, A. L., and Friesen, J. D. (1984) Proc Nd1 Acnd Sci U S A 81(7), 2157-61 Allison, L. A., Moyle, M., Shales, M., and Ingles, C. J. (1985) Cc11 42(2), 599-610 Sweetser, D., Nonet, M., and Young, R. A. (1987) Proceediqs of the Nntiortnl Acndeviy of Scierices of the United Stntes of Antericn 84(5), 1192-6 Kolodziej, P., and Young, R. A. (1989) Mokcirlar & Cellirlnr Biology 9(12), 5387-94 Edwards, A. M., Kane, C. M., Young, R. A., and Komberg, R. D. (1991) 1 Bi01 Clietti 266(1), 71-5 Woychik, N. A., and Young, R. A. (1989) Molecdnr Cellitlnr Biology 9(7), 2854-9 Woychik, N. A., Liao, S. M., Kolodziej, P. A., and Young, R. A. (1990) Gews 6. Deueloprrrertt 4(3), 313-23 Archambault, J., Schappert, K. T., and Friesen, J. D. (1990) Molecrtlar 6. Cellirlar Biology 10(12), 6123-31 Woychik, N. A., and Young, R. A. (1992) Proceedirigs of the Nntiortnl Acndertry of Scierlces of the United Stntes of Anrericn 89(9), 3999-4003 McKiine, K., Richards, K. L., Edwards, A. M., Young, R. A., and Woychik, N,A. (1993) Yenst 9(3), 295-9 Woychik, N. A., Lane, W. S., and Young, R. A. (1991) jortntnl of Biological Clrerrristry 266(28), 19053-5 Woychik, N. A., and Young, R. A. (1990) joiirrtnl of Biologicnl Clie~tiistry265(29), 17816-9 Woychik, N. A., McKune, K., Lane, W. S., and Young, R. A. (1993) Cerie Exyressio~~3(1), 77-82 Treich, I,, Carles, C,, Riva, M., and Sentenac, A. (1992) Getie Expr 2(1), 31-7 Eisenmann, D. M., Dollard, C., and Winston, F. (1989) Cr11 58(6), 1183-91 Hahn, S., Buratowski, S., Sharp, P. A., and Guarente, L. (1989) Ce11 58(6), 1173-81 Hahn, S. (1998) Ce11 95(5), 579-82 Pinto, I.,Ware, D. E., and Hampsey, M. (1992) Ce11 68(5), 977-88 Henry, N. L., Campbell, A. M., Feaver, W. J., Poon, D., Weil, P. A., and Kornberg, R. D. (1994) Certes Dev 8(23), 2868-78 Feaver, W. J., Henry, N. L., Bushnell, D. A., Sayre, M. H., Brickner, J. H., Gileadi, O., and Kornberg, R. D. (1994) 1 Bi01 Clteni 269(44), 27549-53 Gileadi, O., Feaver, W. J., and Kornberg, R. D. (1992) Scierlce 257(5075), 1389-92 Feaver, W. J., Henry, N. L., Wang, Z., Wu, X., Svejstrup, J. Q., Bushnell, D. A., Friedberg, E. C., and Kornberg, R. D. (1997) 1 Bi01 Clierr~272(31), 19319-27 Feaver, W. J., Svejstrup, J. Q., Bardwell, L., Bardwell, A. J., Buratowski, S., Culyas, K. D., Donahue, T. F., Friedberg, E. C., and Komberg, R. D. (1993) Cd75(7), 1379-87 Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Komberg, R. D. (1995) Ce11 80(1), 21-28 Feaver, W. J., Svejstrup, J. Q., Henry, N. L., and Kornberg, R. D. (1994) Ce11 79(6), 1103-1109 Svejstrup, J. Q., Feaver, W. J., and Komberg, R. D. (1996) 1 Bi01 Cherri 271(2), 643-5 Ranish, J. A., Lane, W. S., and Hahn, S. (1992) Scierice 255(5048), 1127-9 Myer, V. E., and Young, R. A. (1998) j Bi01 Chenr 273(43), 27757-60 Table 1.2: Components of the veast RNAP II holoenzvme (vart 1 of 2)
Gene Size Essential Function Gene regulation Interactioiis Structura t Rcferences (kW (Y/N) motifs
SRB componenb SRB2* 23 N template commi hnent SRBQS 78 Y
template commihnent
SrblOp/ 1lp?
cyclin dependent Tiry1 -Ssrr6 Srbllp CTD kinase mediated suppression SrblOp cyclin partner TirpI-Ss~6 SrblOp mediated suppression MED components* NUTl? 130 HO repression MEDl 64 N MED2 48 N MED4 32 Y MED6 33 Y MED7 26 Y MEDB 25 Y MED9/NUT2 21 HO repression MEDlO/CSEZ 21 chromosome segregation leucine zipper MEDI 1/CSE2 12 Table 1.2: Components of the veast RNAP II holoenzvme art 2 of 2)
Gene Size Essential Function In teractions Structural References (kDa) (Y/NI motifs
SWUSNF components S W11 148 S W12/SNF2 194 S W13 93 SNF5 103 SNF6 38 SNFII 19 s Wy59y 59 S Wp6Ip 61 SNFlZ/S Wp73p64 S Wp82p 82 ANCIflFG3 27
Other holoenzyme components (mediating transcription activators and inibitors) CALI1 120 N GAL repression RCRl 123 Y HO repression SlN4/SSN4 111 N CAL and HO repression PGDl 47 N Suppressor of hprl ROX3/SSN7 25 Y
Notes and references to Table 1.2
This Table was adaptcd from (22). ' indicates that this component was identificd as part of the mediator complex (3) " A mutant form of Galllp (called GalllP) interacts with Gal4p (18)
1. Koleske, A. J., and Young, R. A. (1994) Mt tt re 368(6470), 466-9 2. Nonet, M,L., and Young, R. A. (1989) Ge~refics123(4), 715-24 3. Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornbcrg, R. D. (1994) CeII 77(4), 599-608 4. Thompson, C. M., Koleske, A. J., Chao, D. M., and Young, R. A. (1993) Ccll73(7), 1361-75 Notes and referenccs to Table 1.2 (continued)
Koleske, A. J., Buratowski, S., Nonet, M., and Young, R. A. (1992) Cell69(5), 883-94 Koh, S. S., Ansari, A. Z., Ptashne, M., and Young, R. A. (1998) Mo1 Ce11 1(6), 895-904 Piruat, J. I., and Aguilern, A. (1996) Gerietics 143(4), 1533-42 Hengartner, C. J., Thompson, C. M., Zhang, J., Chao, D. M., Liao, S. M., Koleskc, A. J,, Okamura, S., and Young, R. A, (1995) Gnies Dm 9(8), 897-910 Hengartner, C. J., Myer, V. E., Liao, S. M., Wilson, C. J., Koh, S. S., and Young, R. A. (1998) Mol Ce11 2(1), 43-53 Liao, S. M., Zhang, J,, Jeffery, D. A., Koleske, A. J., Thompson, C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J., and Young, R. A. (1995) Natirre 374(6518), 193-196 Myers, L. C., Gustafsson, C. M., Bushnell, D. A., Lui, M., Erdjument-Bromage, H., Tempst, P., and Kornberg, R. D. (1998) Genes Dev 12(1), 45-54 Lee, Y. C., Min, S,, Gim, B. S., and Kim, Y. J. (1997) Mol Cell Biol17(8), 4622-32 Winston, F., and Carlson, M. (1992) Trettds in Gertelics 8(11), 387-91 Treich, I., Cairns, B. R., de los Santos, T., Brewster, E., and Carlson, M. (1995) Mol Ce11 Bi01 15(8), 4240-4248 Peterson, C. L., and Tamkun, J. W. (1995) Trertds Bioclia~iSci 20(4), 143-6 Cairns, B. R., Levinson, R. S., Yamamoto, K, R., and Kornberg, R. D. (1996) Geries DLYU10(17), 2131-44 Cairns, B. R., Henry, N. L,, and Kornberg, R. D. (1996) Mol Ce11 Biol 16(7), 3308-16 Barberis, A., Pearlberg, J., Simkovich, N., Farrell, S., Reinagel, P., Bamdad, C., Sigal, G., and Ptashne, M. (1995) Ce11 81(3), 359-68 Li, Y., Bjorklund, S., Jiang, Y. W., Kim, Y. J., Lane, W. S., Stillman, D. J., and Kornberg, R. D. (1995) Proc Nntl Acad Sci U S A 92(24), 10864-8 Piruat, J. I., Chavez, S,,and Aguilera, A. (1997) Gerietics 147(4), 1585-94 Gustafsson, C. M., Myers, L. C., Li, Y., Redd, M. J,, Lui, M., Erdjument-Bromage, H,,Tempst, P., and Kornberg, R. D. (1997) 1 Biol Clie~ii 272(1), 48-50 Myer, V. E., and Young, R. A. (1998) 1 Biol Clieiii 273(43), 27757-60 complex (PIC) that is transcriptionally competent upon the addition of nucleotides (Buratowski et al., 1989). The protein components of each fraction have been determined and their corresponding genes identified (see Table 1.2 and (Leuther et al., 1996; Myer and Young, 1998; Orphanides et al., 1996)). Each of these factors has a corresponding activity in rnammalian extracts. Together with RNAP 11, the GTFs comprise the basal transcription complex. Initiation of transcription from most promoters requires the TATA-binding protein (TBP of TFIID) which recognizes and binds to the TATA box upstrearn of the transcription-initiation site. The TBP-prornoter complex can be bound by TFILB. TFIIB makes contact with both TBP, RNAP ïI and the promoter and is involved, dong with RNAP II, in detedning the position of the transcription start-site. RNAP II complexed with TFIIF is able to bind the TBP-DNA-TFEB tripartite complex. Binding of TFIIE and TFIIH completes the pre- initiation complex (PIC). An ATP-hydrolysis-dependent step allows a pre-initiation complex helicase activity in TFlM to melt the prornoter DNA and form the open initiation complex. The single stranded, non-coding DNA in this rnelted region is used to direct the addition of nucleotides by RNA polymerase II. Synthesis of the first phosphodiester bond in the nascent pre-mRNA transcript initiates transcription and is followed shonly afterwards by release (or "escape") of the polymerase from the promoter as subsequent nudeotides are added. The polymerase continues the proçessive addition of nucleotides to the growing mRNA as an elongation complex until it pauses or terminates. The transition from the initiation complex to the actively elongating complex is marked by the hyperphosphorylation of the CTD of RNAP II. This phosphorylation is likely carried out by the TFW associated KinZSpJCcl lp cyclin-dependent kinase and probably plays an important role in altering the set of proteins that are associated with the polymerase (see section 1.2 below). Each of the initiation steps can be achieved in vitro by the step-wise addition of transcription factors, RNA polymerase and nucleotides to a DNA template. Specific protein-DNA and protein-protein interactions are formed at each step; these stabilize the complex and/or induce conformational changes that are required for the creation of the pre- initiation complex that is able to accurately initiate transcription. These interactions have been studied in detaii and are not discussed further here (for a review see (Orphanides et ai., 1996)).
The RNAP II holoenzyme. DNA promoter elements upstream of the initiation site are able to bind transcriptional activators that in turn make contact with components of the transcriptional machinery and enhance transcription initiation and/or elongation (reviewed in (Gaudreau et ai., 1998; Hengartner et al., 1995; Keaveney and Stnihl, 1998; Lee and Lis, 1998; McNeil et al., 1998; Parvin and Young, 1998; Ptashne and Gann, 1997)). While the purified GTFs and RNAP II are able to reconstitute promoter-directed transcription in vitro, they are not responsive to transcriptional activators. A megadaiton-sized complex tenned the
"holoenzyme" was purified as the form of RNA polymerase that was able to respond to activators in an in vitro system (see (Koleske and Young, 1994)). In addition to RNAP II, this complex contained TFIIF, TFTIl3, TFIM and a nurnber of previously identified SRB proteins (see below). Addition of purified TFIlE and TBP to the holoenzyme was able to reconstitute promoter-specific transcription in vitro that was responsive to transcriptional activators.
Although transcription can be reconstituted in vitro in the step-wise fashion described above (Buratowski et al., 1991), this mode1 has recently been replaced by one which proposes that most of the transcription factors dong with RNAP II are recmited to the prornoter in the holoenzyme form. Since two of the components of the yeast holoenzyme (Srb4p and Srb6p) are required for the transcription of most RNAP II genes in vivo (Thompson and Young, 1995) and since they are not found free of the holoenzyme complex (Koleske and Young, 1994). it is thought that the RNAP II holoenzyme is the
dominant form of polyrnerase recruited to promoters in vivo. The individual protein components of the yeast holoenzyme are listed in Table 1.2 except for those components which are also a part of the basal transcription machinery
(Table 1.1). The GTFs associated with the holoenzyme Vary dependiag on the method of purification and the organism from which it is purified (Myer and Young, 1998). The holoenzyme may include only TFIIF, or some or al1 of the GTFs (Greenblatt, 1997; Kim et al., 1994; Koleske and Young, 1994; Ossipow et al., 1995; Pan et al., 1997). The transcription factor TFIIA and the TAFs (TBP-associated factors) are not associated with
the yeast holoenzyme nor are they required for transcription in vitro or from most promoters in vivo (Lee and Young, 1998; Moqtaderi et al., 1996; Walker et al., 1996). The transcription factor TFIIS is another potential component of the holoenzyme (Pan et al., 1997) that is important in transcriptional elongation. The other holoenzyme components fa11 into three categories depending on how they were fust identified and are discussed separately below. One of these, SRBS, was isolated as a synthetic-lethal allele in the research reported in this thesis.
SRBs The yeast SRB proteins were first identified as suppressors of the cold-sensitive phenotype conferred by a partial deletion of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Mutants that retain IO to 12 repeats are cold-sensitive andor temperature-sensitive and mutants with fewer than ten repeats are inviable (Nonet and Young, 1989). SRB2, 4, 5, and 6 were identified as dominant suppressors of the cold-sensitive phenotype (Nonet and Young, 1989; Thompson et al., 1993). Their gene products co- purified with RNAP LI (Thompson et ai.. 1993). Of these, SRB2 and SRBS are non- essential genes but are required for efficient promoter-directed basal transcription activity in yeast extracts and appear to play a role in template-cornmitment (Thompson et al., 1993). In cornparison, SRB4 and SRBa are essentiai genes and are required for the majority of RNAP II-directed transcription in vivo (Thompson and Young, 1995). Affinity chromatography and yeast two-hybnd assay s have been used to detect pairwise interactions among the four proteins and establish them as a subcomplex within the holoenzyme (Koh et al., 1998). In addition, Srb4p has been shown to be a direct target of the Gal4p transcriptional activator (Koh et al., 1998). SRB8 to II were identified as recessive suppressors of the CTD tmncation. Each of these was identified previously as SSNS, SSN2, SSN3/UMES and SSN8 in genetic screens for negative regulators of SUC2 and SP013 transcription (Kuchin et al., 1995; Song et al., 1996; Surosky et al., 1994). These genes are essentiai for the repression of a wide variety of genes including the GAL genes that are involved in galactose rnetabolism and that require SNFl for their derepression (Carlson, 1998). SRBlO and SRBIl encode a cyclin-dependent kinase activity that phosphorylates the CTD (Hengartner et al., 1998; Liao et al., 1995). This CTD-kinase activity differs from that found in TFIIH (KIN28) in that it represses transcriptionai activity by phosphorylating the CïD before formation of the pre-initiation complex whereas KIN28 activity occurs after pre-initiation complex formation to convert RNAP II to the elongation-comptent form (see below).
GAL.1 1, SIN4, RGRI, ROX3, and PGDI/HRSI/MED3 These components of the holoenzyme fa11 into a single class of factors that probably act gericrally to repress trwrscnption and were identifïed previously in severai different genetic scxens (reviewed in (Myer and Young, 1998)). Deletions in Rgrlp cause Gd1 lp, Sin4p and Pgdlp to dissociate from the holoenzyme, suggesting that their common function in vivo may be related to a mediator subsrmcture (Li et al., 1995). HoIoenzyme lacking these three subunits remains able to mediate transcriptional activation in vitro using purified transcription factors. These resdts dong with the identification of SWOand SRBI 1 are consistent with the idea that holoenzyme components may also mediate the effects of trinscriptional repressors as well as transcriptional activators in vivo.
Componenrs of the mediator (MEDs) A search for biochernical fractions able to mediate the effect of transcription activators resulted in the isolation of a megadalton-size complex termed 'the mediator' (Kim et al., 1994). Components of the mediator included previously identified holoenzyme proteins as well as a new set of proteins called Meds. AU of the mediator components are identified by an asterisk (*) in Table 1.2. Med6p has ken shown to mediate contact between the Med proteins and the Srb2,4,5,6 subcomplex via an interaction with Srb4p (Lee and Young, 1998). Med6p is required for the activation of some genes in vivo and for activation of transcription in vitro (Lee et al., 1997).
S ?VI/SN F Swi/Snf is an eleven-protein subcomplex of the yeast holoenzyme (Wilson et al., 1996) that has chromatin-remodeling activity (Cote et al., 1994). This activity is thought to relieve chromatin-mediated repression of transcription in vivo (Peterson and Tamkun, 1995; Winston and Carlson, 1992). The Swi/Snf complex does not appear in some preparations of yeast holoenzyme (Cairns et al., 1996) and homologues of its subunits do not appear in the human holoenzyme (Pan et al., 1997). Another complex in yeast with the capacity to srnodel the gtructure of çhromatin, termed RSC, is about ten-fold more abundant than the Swi/Snfcomplex (Cairns et al., 1996). Formafion of the ho10ens)me is dependent un the CTD Interaction of the mediator complex with RNA polymerase appars to be dependent on the CTD of the RNAP II largest subunit (Rpblp in yeast). Antibodies directed against the non-phosphorylated form of the CTD were able to displace mediator from core RNAP II, indicating that anti-CTD antibody (8WG16) and mediator probably compete for a comrnon target (Kim et al., 1994). Furthemore, the Srb2,4,5,6 protein subcomplex was retained on a CID column dong with TBP and at least a dozen other proteins. Proteins retained by the CTD column did not include subunits of RNAP II, suggesting that column-bound CïD was able to compete with RNAP II in a yeast extract for at least a portion of the mediator complex (Thompson et al., 1993) and TBP (Usheva et al., 1992). The emerging picture, in sumrnary, from the studies described above is as follows: the effects of positive and negative transcriptional regulatoa on RNAP Ii core activity is mediated by a large complex of proteins and this complex is associated with the polymerase via the CTD. This is consistent with the fact that the CTD is required for response to transcriptional activators/enhancers in vivo for both yeast and mammals (Allison and Ingles, 1989; Gerber et al., 1995; Scafe et al., 1990) and that CTD phosphorylation is not required for basal level transcription (Makela et al., 1995; Serizawa et al., 1993).
1.2 Transition from the pre-initiation complex to the elongation compiex
Af'ter the holoenzyme has bound to the promoter and fonned the open complex, RNA polymerase is abte to form the first bond between the first two nucleotides of the pre- rnRNA. The RNA polymerase continues to add nucleotides and eventualiy leaves the region of the promoter and enters into the elongation phase of transcription. This transition or "promoter escape" is marked by at least three changes to the complex that initiated transcription. First, the CTD becomes hyperphosphorylated. Second, al1 of the GTFs (except for TFIIF) dissociate from the RNAP II complex (Zawel et al., 1995). Third, other proteins specific to the RNAP II elongation cornplex become associated with the polymerase (Otero et al., 1999). Each of these changes requires that the elongation complex be quite different from the initiating complex. While the composition of the elongating complex is not well-defmed, the phosphorylation of the CTD has ken studied in detail and is central to the processes of mRNA biogenesis.
Phosphorylation of the CTD
A number of kinases can phosphorylate the CTD. The kinase which is most relevant to the process of transcription is the Kin28p subunit of TFIM and its associated cyclin Cc1 lp that phosphorylates serines at position 5 of the CTD repeat (YSPTSPS) (Cismowski et al., 1995; Feaver et al., 1994). The mamrnalian TFIM subunit homologues cdk7M015 and cycH possess an equivalent cyclin-dependent kinase activity (Roy et al., 1994; Serizawa et al., 1995; Shiekhattar et al., 1995) . This activity is enhanced in mammalian cells by the p56 subunit of TFIIE (Serizawa et al., 1994). In addition, the CTD may be phosphorylated on both Ser2 and Sers by recombinant human cdc2kyclin B (Gebara et al., 1997).
The hyperphosphorylated CTD is associated with elongation Hyperphosphorylation of the mammalian CTD is associated with a transition from the initiation-comptent RNAP II to the productive elongation complex (Cadena and Dahmus,
1987; Chesnut et ai., 1992; Dahmus, 1996; Dahmus, 1995; Dahmus, 1996; Dahmus, 1994; Laybourn and Dahmus, 1990; Payne et al., 1989; Weeks et al., 1993). This property has ken most elegantly demonstrated in a series of experirnents in Drosophila.
The uninduced Drosophila hsp70 gene contains an RNA polymerase il complex that pauses in the %end of the gene after synthesizing a short transcript (O'Brien and Lis, 1991). DNA associated with this complex can be detected by DNA-blot analysis after immunoprecipitation from a W cross-linked extract with antibodies to the CD.DNA corresponding to the 5'-portion of the gene is CO-immunoprecipitatedby antibodies against the non-phosphorylated CTD, but not the phosphorylated CTD in uninduced cells.
However, in induced cells both antibodies are able to immunoprecipitate this DNA as well as DNA corresponding to the 3'-portion of the gene. This indicates that phosphorylation of the CTD is associated with the transition of polymerase from a paused to an eIongating form (O'Brien et al., 1994).
Phosphorylation of the CTD is most likely associated with changes in the protein population that accompanies elongating RNAP II. Many of the holoenzyme components are associated with the CTD (see above) and changing the charge density of this structure might alter these associations. This possibility is highlighted by the fact that phosphorylation causes a global conformational change in the CTD as detected by differences between CTDo (hyperphosphorylated) and CTDa (hypophosphorylated) sedimentation rates on sucrose gradients and mobility in gel filtration chromatograms
(Zhang and Corden, 199 1). 1.3 Initiation of transcription and the biogenesis of mRNA
The C-terminal domain (CT'D)of the largest subunit of RNA polymerase II plays a pivotal role in coordinating many of the processes involved in mRNA biogenesis. As described above ,the CTD is involved in stabilizing interactions between the core RNAP II and the rest of the holoenzyme. in addition, the CTD appears to be phosphorylated sometime before or shortly after passage of the polymerase into the elongation phase. Finaiiy, the CTD plays a role in coordinating factors that act subsequent to initiation. For example, the CTD cm bind to factors required for capping, efficient splicing and 3'- cleavage of the nascent transcript. It is unclear when these proteins become associated with the polymerase. So fa, the earliest-recruited factors are the marnmalian cleavage and polyadenylation stimulatory factor proteins (CPSF) that are probably associated with the holoenzyme as it initiates transcription (Dantonel et al., 1997). In addition, the capping enzymes specifically bind to the phosphorylated form of the CTD and thus they probably join the polymerase complex shortly after transcription is initiated (McCracken et al., 1997). The association of these proteins with the polymerase has encouraged the idea that mRNA is made in "factory-like" structures chat coordinate transcription, capping, splicing and 3'-processing of nascent transcnpts (McCracken et ai., 1997). It is possible that the nascent mRNA is capped as soon as the polymerase is committed to the elongation phase of transcription. Other mechanisms associated with the polymerase may irnmediately transfer the nascent message to a nearby spliceosome complex for removal of introns as transcription continues. Other factors associated with the elongating polymerase may recognize and bind to the 3'-processing site in the nascent rnRNA, which in tum could trigger transcription termination, 3'end cleavage and polyadenylation of the pre-rnRNA.
Evidence to support each of these ideas will be discussed below. 2.1 5'-end capping of pre-mRNA
Eukaryotic RNA polymerase U transcripts are modified CO-transcnptionally by the addition of a 5'-methylated guanosine cap structure at their S'-end (reviewed in (Shuman,
1995)). This cap structure is unique to RNAP II transcripts; neither RNAP 1 transcripts (ribosomal RNAs) nor RNAP III transcnpts (tRNAs) are capped. Capping is carried out by three enzymatic activities. First, the 5'-most phosphate of the pre-mRNA is removed by a RNA triphosphatase. Second, an RNA guanylyltransferase reacts with GTP and forms a covalent GMP-guanylyltransferase intermediate. The GMP is then transferred to the 5'- diphosphate end of the pre-mRNA via a 5'-to 5'-phosphate linkage. Third, the guanine cap is methylated at the N7 position by an RNA (guanine-7)-methyltransferase to complete the rn7~(5')~~~(5')~cap structure. The triphosphatase and the guanylyltransferase activities are contained in a single bi- functional peptide in multicellular organisrns, while in yeast these activities are carried out by separate proteins that CO-puri@as a stable complex. The methyltransferase is a separate protein in both cases. The yeast capping enzymes are encoded by three essential genes; CETI(PRP34) encodes the RNA 5'-triphosphatase (Tsukamoto et al., 1997), CEGI(PRP33) encodes the guanylyltransferase (Schwer and Shuman, 1994) and ABDI encodes the methyltransferase (Mao et al., 1995) (see Table 1.3). The cap is bound by a cap-binding complex (CBC) that is composed of two proteins (CBP20 and CBP80 in humans or the respective orthologues Mudl3p and Gcr3p in yeast). Neither of the yeast cap-binding proteins is essentiai. An ailele of GCR3 was isolated as a synthetic-lethal in this thesis. Figure 1.3: The steps involveci in capping the S'-end of nascent pre-mRNA.
The three enzymatic steps required to cap pre-mRNA are shown in panel A (see text for details). The letters "pppN"in the first iine represent the S'-end of the pre-mRNA that is to be capped. A schematic of the 5'-cap structure is shown below in panel B. Note that position N7 of the guanosine base is methylated. The capping enzymes are listed in Table 1.3. AdoMet is S-adenosylmethionine and AdoHcy is S-Adenosylhomocy steine. A. The three steps required to form the 5'-cap structure
ppN .... + Pi
G(S)papS'pa'N ... + PPi
methylytransferase G(5')p&ppalN-..+ AdoMet > m7~(~)papppa*~...+ AdoHq
B. The s'-cap structure (m7~(5')papSpd~. ..)
N... Table 1.3 Capping enzymes and cap-binding complex of yeast
Gene Size Essential Description Interactions References (kDa) W/N)
GPP~~enzymes CETlPRP34 62 Y RNA 5'- triphosphatase Ceglp (1-3) CEGlPRP33 53 Y guanyly ltransfer ase Cetlp, CTD (1,3,4) ABD1 50 Y methyItransferase CTD (3,5)
Cap-binding complex (CBC) GCR3/STOI 99 N largest subunit Mudl3p. srplp§ (6.7) CBP80 orthologue (17%) MUD13 23 N mallest subunit Ga3psrplps (8) CBP20 orthologue (51%)
Notes and References to Table 13
Srpl p is equivalent to yeast irnportin a
Cho, E. J., Rodriguez, C. R, Takagi, T., and Buratowski, S. (1998) Genes Deu 12(22), 3482-7 Tsukamoto, T., Shibagaki, Y., imajoh, 0.S., Murakoshi, T., Suzuki, M., Nakamura, A., Goroh, H., and Mizumoto, K. (1997) Biochem Biophys Res Commiin 239(1), 116-22 McGacken, S., Fong, N., Rosonina, E., Yankdov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Shuman, S., and Bentley, D. L. (1997) Genes Dm 11(24), 3306-18 Shibagaki, Y., Itoh, N., Yamada, H., Nagata, S., and Mizurnoto, K. (1992) J Biol Chem 267(14), 9521-8 Mao, X., Schwer, B., and Shuman, S. (1995) Mol Celf Biol15(8), 4167-74 Goriich, D., Kraft, R., Kostka, S., Vogel, F., Hartmann, E., Laskey, R. A., Mattaj, 1. W., and Izaurraide, E. (1996) Ce11 87(1), 21-32 Uemura, Fi., and Jigami, Y. (1992) J Bacteriof 174(17), 5526-32 Colot, H. V., Stutz, F., and Rosbash, M. (1996) Genes Dm 10(13), 1699-708 2.2 Capping and mRNA biogenesis
The addition of the cap to nascent pre-rnRNAs is important to each of the events that subsequently take place in the lifetime of the RNA. The cap and CBC have been implicated (directly or indirectly) in efficient splicing in the mammalian and yeast systems (Edexy and Sonenberg, 1985; Konarska et al., 1984; Lewis et al., 1996a), in marnmalian 3kleavage of the pre-rnRNA (Flaherty et al., 1997; Gilmartin et al., 1988), in RNA export in higher eukaryotes (Gorlich et al., 1996; Hamm and Mattaj, 1990), in efficient translation in al1 eukaryotes (Shatkin, 1985) and in protecting mRNA from 5'-3' exonucleases in al1 eukaryotes (reviewed in (Beelman and Parker, 1995; Schwer et ai., 1998)). 1 will discuss most of these roles separately below in the relevant scctions. In this section, 1 will limit my comments to the relationship between capping and transcription initiation.
Cuppirrg occurs CO-~anscriptionafly There is evidence from studies with Drosophila RNA polyrnerase II that transcripts are capped soon after initiation has occurred. The Drosophila RNA polymerase pauses at promoter proximal positions between +17 and +37 on the uninduced prornoter of the hsp70 gene and between +28 to +46 on the uninduced promoter of the hsp27 gene. For both genes, nascent transcnpts appear to be capped before reaching 20 nucleotides in length; the majority of transcripts are capped by the time they reach a length of 30 nucleotides. (Rasmussen and Lis, 1993). Co-transcriptional capping has also been observed in the case of the vaccinia-virus capping enzyme, which binds directly to the viral RNA polymerase and caps nascent transcripts by the tirne they are at least 31 nucleotides in length (Hagler and Shuman, 1992). One would expect that a nascent pre-mRNA could not be capped until it has been extmded from the RNA-binding pocket of the polymerase. This last result is consistent with the size of the pocket (18 nucleotides. in length) as detemiined by nbonuclease footprinting of nascent messenger RNA within ternary complexes (Hagler and Shuman, 1993). Alîhough not demonstrated, CO-transcriptionalcapping in yeast seems likely given
the conserved nature of the systems involved (see below).
Capping enzymes are recruited tu RNAP II by the CTD The guanylyltransferase capping enzyme is able to bind directiy to the phosphorylated (but not the non-phosphorylated) CTD in both the yeast and mammalian systems (Cho et al., 1997; McCracken et al., 1997). In addition, the yeast rnethyltransferase is also able directly and independently to bind the phosphorylated form of the CTD (McCracken et al., 1997). The yeast triphosphatase binds indirectly to the CTD via the guanylyltransferase (Cho et al., 1998). The specificity for the phosphorylated CTD helps to explain a number of previous
observations. First, the fact that the CTD does not have a counterpart in either RNAP 1 or RNAP ïIi explains why only pre-mRNAs have a m7G cap and provides a mechanism by
which these RNAs cm be targeted to the subsequent events specific to capped RNAP II transcripts. Second, the presence of the capping enzyme in the RNAP II complex explains how transcripts can be capped CO-transcnptionallyand the fact that this presence is dependent on the phosphorylated form of the CTD suggests that only those RNAP II complexes which have cornmitted to the elongation phase will be able to cap nascent pre- mRNAs (O'Brien et al., 1994). The interaction between capping enzymes and the CTD was shown to be significant biochemically since two different pre-mRNA transcripts (HIVZ CAT and p-globin)
synthesized in vivo using an RNA-polymerase with a truncated CTD (A5) were less- efficiently capped at their S'-ends than transcripts made by wild-type (WT) RNAP II (McCracken et al., 1997). This effect was independent of the presence of an intron or a WT poly(A) site in the transcript indicating that the reduction in capped transcripts was a direct result of the CTD deletion and not indirectly related to poor splicing or 3'-end processing . The observation that capping can and does occur shortiy afier transcription initiation raises the interesting possibility that capping rnight play a role in the efficient transition to the elongation phase (Jove and Madey, 1982). Since neither the cap nor the capping enzymes are required for transcription in an in vitro reconstituted system, capping may only play an auxiiiary role or oçcur entirely as a consecpence of the transition to the elongation phase. ln either case, capping ensures that transcripts are protected against 5'-3' exonucleases and will be targeted efficiently to subsequent processing events. 3.1 Splicing
Removd of non-coding sequences (introns) from pre-mRNAs occurs as a two-step reaction. Three cis-elements containeci in the intron are required for splicing. These include the 5'-splice site (consensus GUAUGU), the 3'-splice site (consensus YAG) and the branchpoint sequence in the intron (consensus UACU-; the "branchpoint" adenosine is underlined). The first step of splicing involves a transesterification in which the 2' hydroxyi group of the 'branchpoint' adenosine attacks the phosphate group of the first intron nucleotide. This step results in a free hydroxyl group at the 3'-end of the fust exon
and an intron loop structure (Iatiat) that is stïii bound to the second exon. The second step of splicing involves a transesterification between the free 3'-hydroxyl group of exon- 1 and
the first phosphate of the exon-2. The resulting end products are the two exons spliced together and the released lariat intron. These basic steps are conserved among yeast and mammals. Uniike transcription, eukaryotic splicing of introns from pre-rnRNA has not been reconstituted in vitro using purified components. However, the splicing reaction has been studied in detail in both mammalian and yeast systems using celi-free extracts and pre- mRNA transcripts made by bacteriophage RNA polyrnerases. The splicing reaction requires five U-rich, small nuclear RNAs (U snRNAs; Ul, U2, U4, U5 and U6) that in turn are complexed with proteins to form the five smaiï nuclear ribonucleoprotein particles (U snRNPs). These snRNPs assemble together with non- snRNP proteins on the pre-mRNA in a step-wise manner to form the active spliceosome and each of the snRNPs play a distinct role in this process (reviewed in (Xu, 1998)). Many of the steps in spliceosome assembly and the components involved at each step have been defined by assaying the formation of the intermediates and products of the splicing reaction in vitro. For example, in yeast, the pre-mRNA substrate is committed to the splicing pathway by an early 'cornmitment' complex that forms in the absence of ATP (Seraphin and Rosbash, 1989). This complex involves interactions between the U1 snRNP, the 5'-splice site, Mud2p and the branchpoint sequence. A similar 'early' complex forms in the mamrnaiian system (Michaud and Reed, 1991). In both systems, the addition of ATP allows formation of 'mature' spliceosorne complexes containing U2, U4N6 and the US snWsin which the two tramesterification steps of splicing rnay occur (reviewed in (Moore et al., 1993)). Early and late splicing complexes in both yeast and mammalian systems may be distinguished by their rnobility in non-denaturing gel systems and this method has been central to determining the role of splicing factors in spliceosome assembly. Non-snRNP proteins also play important roles in splicing. Examples of these include the branch-point binding protein (SFI/BBP) and senne/arginine repeat (SR) proteins such as U2AF65 in the human system. Classical SR proteins have a repeated serine-arginine motif as well as one or two RNA-recognition motifs (RRMs) and function by promoting spliceosorne formation in higher eukaryotes as well as by regulating the use of alternative splice sites (reviewed in (Fu, 1995)). No typical SR proteins have been reported in the yeast Saccharomyces cerevisiae and only one has been reported in the fission yeast Schizosaccharomyces pornbe (Gross et ai., 1998).
3.2 Splicing and mRNA biogenesis
Splicing in higher eukaryotes occurs CO-tramcriptionallyand CO-localizeswith transcription complexes. A number of studies using electron rnicroscopy of chromatin spreads from higher eukaryotes have provided evidence that the splicing of many introns occurs co- transcriptionally (Beyer and Osheim, 1988; Beyer and Osheim. 1991). For example, in C. tetans, the Balbiani ring pre-mRNAsl introns are generaiiy spliced co-transcnptionally in a 5'-3' order, although some introns are spliced post-transcriptionally depending on their position in the gene (Bauren and Wieslander, 1994; Wetterberg et al., 1996). In addition, isolation and S 1 nuclease analysis of nascent transcnpts associated with a chromatin pellet from mammalian ceiis indicate that splicing occun both CO-transctiptionallyas well as post- transcriptionally (Wuarin and Schibler, 1994). No direct evidence exists at this point suggesting that splicing is co-transcriptional in yeast (Elliott and Rosbash, 1996). A number of antibodies that recognize either snRNPs or SR proteins have ken used in situ to show that mammalian splicing components are loçalized to discrete domains in the nucleus called speckles (Blencowe et al., 1994; Nickerson et al., 1995; Spector, 1993). More recently, RNAP II has also been iocalized to speckie domains using antibodies 83, CC-3, H5 and HI4 that specifically recognize the hyperphosphorylated CTD (Bisotto et al., 1995; Bregman et al., 1995; Mortillaro et al., 1996; Vincent et al., 1996). The co- localization of splicing components with RNAP II appears to be biologically relevant since antibody CC-3 is able to inhibit splicing in vitro, co-imrnunoprecipitate pre-rnRNA splicing intermediates, lariat-intron and mature mRNA as weil as the U-rich snRNPs (Chabot et al., 1995). In addition, anti-snRNP antibodies or anti-SR protein antibodies are able to co- immunoprecipitate the hyperphosphorylâted form of RNAP II but not the hypophosphorylated form (Kim et al., 1997; Mortillaro et al., 1996). These observations are consistent with the idea of CO-transcriptionalsplicing, since one would expect the elongating form of polymerase to be associated with splicing factors. However, this co- imrnunoprecipitation of hyperphosphorylated RNAP II occurs even in the absence of pre- mRNA or transcriptional activity (Kim et ai., 1997). Furthemore, speckles defined by splicing proteins and/or hyperphosphorylated RNAP II are large and well-defined in transcnptionally inactive cells, but disperse into imegdarly-shaped speckles intercomected by a reticular network in transcriptionally active cells (Bregman et al., 1995). One explanation is that phosphorylation of the CTD is an indication of its association with splicing related factors and that the RNAP II present in these complexes may or may not be transcriptionally engaged. The CTD and splicing More evidence that the CTD might couple splicing and transcription in higher eukqotes was provided by the observation that the CTD peptide is able to inhibit splicing in HeLa extracts (Yuryev et al., 1996). Furthemore, CTD expressed in vivo is also able to inhibit splicing in higher eukaryotes and this activity is associated with the ability of the expressed CTD to disperse splicing factors that are normally associated with nuclear speckles (Kim et al., 1997). These results are not easily explained by a mode1 in which the CTD simply couples splicing components to the transcriptional machinery. Somehow the CTD must aiso be able to disrupt interactions between splicing factors that are normally juxtaposed and thereby decrease the efficiency with which splicing occurs. in any case these results suggest that the CTD may interact directly with at least some splicing factors as shown before. The hypothesis that splicing rnight be coupled to transcription through the C-terminal domain of RNA polymerase II was fust raised by Greenleaf well before any supporting physical evidence existed (Greenleaf, 1993) . Greenleaf proposed that the two processes rnight be coupled through an interaction between the positively charged domains of SR proteins and the negatively charged phosphorylated CID of RNAP II (recently reviewed in (Corden and Patturajan, 1997; Steinmetz, 1997)). SR proteins that interact with the CTD have been found, however the CTD-interacting domains of these proteins are, in most cases, separate from the SR domains (Corden and Patturajan, 1997). A number of SR-like proteins that interact with the CTD were isolated using the yeast two-hybrid system. These inciude the mouse proteins niAl, mA9, mA4, mA8 and their related rat proteins rAl, rA9, rA4 and rA8. The rnouse proteins were isolated in a two- hybnd screen (Yuryev et al., 1996) using the ldmost distal mouse CTD repeats which are functional in the context of yeast RNA polymerase II but do not activate transcription by themselves when used as bait. The mouse clones from the two-hybrid assay were used as probes to clone the full-length rat proteins from a genomic library. Each of the rat proteins
was also able to interact with the CTD in a two-hybrid assay and sequence analysis revealed the presence of arginine-serine (SR) repeats in each. Potential RRMs were identified in only two of the proteins (rA8 and rA4); thus, only these two can be considered SR-like proteins. Deletion analysis revealed that the CTD-binding domains of these proteins fa11 into two groups; rAl and rA9 have similar 80 amino acid C-terminal sequences that are responsible for binding the C'ID while rA8 and rA4 have simila. 120 amino acid N- terminal domains that are required for this function in two-hybrid assays. Recombinant GST-rA1 protein was shown to interact directly with both phosphorylated and non- phosphorylated yeast RNAP II and this binding could be partially competed for by WT
CTD peptide. A requirement for phosphorylation of the CTD could not be mled out since the polymerase DA was phosphorylated to sub-stoichiometric arnounts. It has not been demonstrated that any of these proteins plays a role in splicing. The yeast protein Nrdlp was identified by sequence similarity to the rat proteins rA8 and rA4. Nrdlp is a sequence- specific RNA-binding protein and its N-terminal domain is able to interact with the CïD in a two-hybrid assay (Yuryev et al., 1996)). It probably plays a role in elongation and is discussed below in section 4.2. SRcyp/CASPlO (serine/arginine-rich cyclophilin or CTD-associated SR-like protein) was isolated from a marnmdian Library in a two-hybrid screen using the fuil-length mouse CTD (Bourquin et al., 1997). A C-terminal domain (arnino acids 520 to 667) that is rich in
SR dipeptides ( 13% between amino acids 540 and 667) is both essential and suffkient for binding to the RNAP II CTD in a two-hybnd assay. It has not been determined whether this interaction is a direct one. Although CASPlO does not have a demonstrated role in splicing, it was shown to localize to nuclear speck!es containing splicing factors. In addition. this protein undergoes SC35-Wre redistributicn upon inhibition of transcription. Other, non-SR proteins that interact with the CTD have been found. PSF and p54nrb were both detected as proteins capable of specific binding to imrnobilized, full-length mouse-CTD (Emili, 1997). PSF is an essential rnammalian splicing factor that crosslinks to RNA in splicing complex-C. Extracts that are immuno-depleted of PSF show a step II- splicing defect (Gozani et al.. 1994). The role of p54nrb in splicing is unknown although it shares 71% identity with PSF over a 320 arnino acid stretch (Dong et al., 1993). PSF binds to p54nrb and together these proteins CO-puri@with DNA topoisomerase I in a 1: 1 : 1 complex (Straub et ai., 1998). The presence of PSF and p54nrb enhance DNA topoisomerase activity, aithough it is not known what hinction this plays in splicing. fnterestingly, DNA topoisomerase 1 has a kinase activity that is specific to SR-proteins in mamrnalian ceils (Rossi et al., 1996). In conclusion, (rAl) has been shown to interact directly with the CTD but it is unclear what role it plays (if any) in splicing. Furthemore, PSF interacts with the CTD and has a demonstrated role in spücing but it is not known how these two acuvities are related. More research is required to estabLish the nature of interactions between the CïD and components of the spliceosome. Again, there is no evidence that the CTD couples splicing and transcription in yeast. This issue is addressed in Chapter 4 of the thesis.
The cap and cap-binding complex are required for eficient splicing Both the cap and the cap-binding complex are required for efficient splicing in rnamrnalian systems. Pre-mRNA substrates with a 5'-end m7~ppp~cap are efficiently spliced in HeLa extracts (Edery and Sonenberg. 1985; Konarska et al., 1984). In addition, m7~cap analogs added to splicing extracts inhibit splicing activity when added at the start of the reaction but not if added at later times indicating that the cap affects an early step in splicing. Capped pre--A is bound in mamrnaiian systems by CBC which is composed of two proteins (CBPBO and CBPîO). HeLa extracts that are immunodepleted of CBC are also inhibited at an early step in the splicing process. No intermediate products are accumulated and a decrease is observed in al1 three splicing complexes that iead to the
mature spliceosome (A, B and C) (Izaurralde et al., 1994). The early step facilitated by the cap and the CBC is the association of the U1 snRNP with the cap-proximal 5'-splice site (Lewis et al., 1996a). This step does not require the presence of ATP so formation of the early complex at the 5'-splice site (E5')was exarnined in the absence of ATP. Formation was shown to be reduced by 50% in splicing extracts that were immuno-depleted of CBC compared to those that were mock-depleted (Lewis et al., 1996a). The ES1-complex consists of contacts made between the U1 snRNP and the 5'-splice site, suggesting that formation of these bonds might be facilitated by CBC. This
hypothesis was supported by experiments which demonstrated that efficient psoralen- mediated cross-linking of U 1 snRNP to the 5'-splice site was dependent on the presence of both a 5'-rn7~mRNA cap and a CBC complex (Lewis et al., 1996a). Efficient Ul cross- linking could be restored by adding back recombinant CBC purified from E. coli, indicating that CBC was responsible for the observed effect in the immuno-depleted extract. However, this effect could not be replicated using purified Ul snRNP and CBC components, indicating that additional factors are required to mediate the effect of the CBC on ES-cornplex formation. These additional factors are unknown. Other studies have suggested that the cap is not required for efficient U 1-snRNP-dependent complex assembly at 5'-splice sites but is instead required for the replacement of U1 snRNA at the 5'-spiice site by U6 snRNA (O'Mullane and Eperon, 1998). Unlike splicing in HeLa extracts, yeast pre-mRNA substrates do not have to be capped in order to be spliced efficiently by yeast extracts and cap anaiogs are not inhibitory to splicing activity (Lin et al., 1985). However, studies with conditional mutations in the guanylyltransferase subunit (Ceglp) of the capping enzyme showed that capping is important for efficient splicing in vivo, even though it is not absolutely required (Fresco and Buratowski, 1996; Schwer and Shuman, 1996). Conditional mutations in this gene were unaffected for both polyadenylation and nuclear export of mRNA (Fresco and Buratowski, 1996).
The yeast CBC plays a role similar to the human CBC in splicing. The yeast equivalents of the CBP20/80 subunits of CBC are Mudl3p and Gcr3p. respectively (Gorlich et al., 1996). MUD13 was first identified in a screen for mutations that are syntheticaily-lethal with a viable mutant dele of U 1 snRNA (Colot et al., 1996) and GCR3 was identified by its sequence similarity to CBP80. Extracts lacking Mudl3p or Unmunodepleted of CBC are deficient in cbeir abiüty to form commitment complexes with pre-mRNA aithough the effect on splicing efficiency is less pronounced (Colot et al.. 1996; Lewis et al.. 1996b). The foxmation of the commitment complex is equivalent to that of the early complex in mammalian cells and is also ATP-independent. The addition of purified yeast CBC to CBC-depleted extracts restores cornmitment-complex formation, indicating that it may play a similat mle in splicing as dœs its human counterpart. 4.1 Processing of the pre-mRNA 3'-end
Yeast pre-mRNAs are processed at their 3'ends to yield transçripts that have a poly(A) tail of about 50-70 adenosine residues. This processing event proceeds in two steps; ht, the nascent pre-mRNA is cleaved upstream of its 3'-end and then the poly(A) tail is added to the 5'-cleavage product (reviewed in (Colgan and Maniey, 1997; Keller and Minvielle- Sebastia, 1997). Two pre-mRNA cis-acting elements ups~of the cleavage site (Py(Ah) are required for 3'-processing in yeast; an A-rich positioning element determines the position of the 3'- cleavage site and further upstream an AU-rïch efficiency element acts to enhance the eff~ciencyof the positioning element (Guo and Sherman. 1996). The protein elements that carry out the process of 3'-processing are shown in Figure 1.4. Cleavage factors 1 and II (CF IA, CF JB, and CF II) are sufficient for the ATP dependent 3'4eavage while addition of the poly(A) tail requires CF IA, CF IB, poly(A) polymerase (PAP) and polyadenylation factor I (PF 0. Fiplp (a subunit of PF I) tagged with histidines was used to purify a complex that contains previously identified components of PF 1 along with PAP and the four components of CF II and CF IA and CF IB (Preker et al., 1997). This complex is able to polyadenylate a pre-cleaved mRNA without added PAP. Mammalian pre-mRNAs are processed at their 3'ends in a similar two-step pnress to yield transcripts that have longer poly(A) tails that are on average 250 nucleotides in length. (reviewed in (Keller and Minvielle-Sebastia, 1997)). The pre-mRNA cis-acting elements required for mammalian 3'-proçessing include a highly conserved AAUAAA sequence upstream of the cleavage site and a GU-nch or U-nch element downsueam of the cleavage site. 3'-cleavage of mammalian pre-rnRNAs in vitro requires four multi-subunit factors: cleavage and polyadenylation factor (CPSF), cleavage stimulation factor F (CstF). cleavage factors Im and IIm (CF Im and CF &). Poly(A) polymerase (PAP) is also required for efficient cleavage of most pre-mRNAs. Subunits of CPSF bind the upstream
AAUAAA element while a subunit of CstF binds to the GU or U rich elements downstream of the cleavage site. In addition, CF Im rnakes contact with RNA and stabiLizes the CPSF- RNA complex. The factor containing the actual endonucleolytic cleavage activity of the first step is unknown. Mammalian polyadenylation can be reconstituted in vitro with only three factors: CPSF, PAP and nuclear poly(A)-binding protein (PAB II). PAP requires CPSF to tether it specifically to the pre-mRNA 5'-cleavage product from the first step. Poiyadenylation wcurs distributively until the poly(A) tail is at least 10 nucleotides in length. At this point,
PAB II is able to bind the poly(A) tail and stimulate PAP activity making it processive.
PAB II also plays a dein limiting the poly(A) tail length. Figure 1.4: A summary of the components involved in yeast pre-mRNA 3'- processing. Complexes are surrounded by gray iines. The PF 1 and CF U complexes dong with PAP cm be CO-purified by nickel-chromatography using a His-tagged Fipl p. Organization of factors in a complex is hypothetical unless otherwise stated. Demonstrated pairwise interactions are indicated by a black dot in a cornplex and by an arrow between complexes. The position of Rnal5 is based on its homology to the mammalian CstF-64 kDa subunit that binds the GU-rich eiement. The position of Cftl is based on homology to the CPSF- 160 kDa subunit which binds to the AAUAAA element. It should be noted, however, that the positional relationship of factors CF I and PF 1 to the pre-mRNA elements has not been established. This Figure is based on Figures from (Keller and Minvielle-Sebastia, 1997;
Preker et al., 1997). References are in the text and in references therein.
Table 1.4: Components of the 3'-processing machinery in ycast (part 1 of 2)
Gene size esscritial properties Mammalian References (kW (Y/N) Orthologue (% identity)
Cleavage factor IA (CF IAy) complex: cleavage and polyadenylation (1) RNA14 80 Y complex binds to pre-mRNA, involved in poly(A) site choice. CstF-77 kDa (24%) (2,3) RNA15 33 Y ha15 contains RRM and can be cross-linked to RNA. CstF-64 kDa (2t3) PCFl 1 72 Y (4 1 CLPl 50 ? CLPl (26%) (1) PA01 70 Y Pablp is required for polyadenylation processivity. PABII (51%) (5) Major poly(A) protein of yeast has 4 RRM domains. Interacts with Rnal5p and elF4E.
Cleavage factor IB (CF IBy) complex: cleavage and polyadenylation HRPI/NAB4 73 Y Isolated as a suppressor of a ts np13 allele. Has 2 RRM domains. Cross-linking to pre-mRNA requires the (UA)6 polyadenylation. efficiency element. Shuttles between nucleus and cytoplasm.
Cleavage factor II (CF 11~)~:3' cleavage YHH1/CFT1 153 Y YDHl/CFTZ 96 ? ATP-dependent binding of pre-mRNA requires (UA)6 element and sequence downstream of the poly(A) site. BRRSIYSHI 88 Y Plays a role in polyadenylation and possibly 3'-cleavage. CPSF-100 kDa (25%) (8-10) Has 53% identity to CPSF-73 over first 500 amino acids. CPÇF-73 kDa (53%) (over 1st 500 a.aJ ? 90 ? same as PTA 1 ?
Cleavage-stimulatory fator: Stimulates cleavage REFZ 59 N Stimulates cleavage at weak poly(A) sites
Poly(A) polymera8e1: Synthesis of poly(A) tail PAPl 64 Y Has 47% identity to mammalian PAP over first 2/3 of protein. Not required for mRNA 3'-cleavage, in contrast to its mammalian homologue. Table 1.4: Components of the 3'-processing rnachinery in yeast (part 2 of 2)
Gene size essential properties Mammalian Referenccs @Da) (Y/N) Ort hologue f% identitvl
Polyadenylation factor complex (PF 1)l: Polyadenylation FIPl 36 Y Interacts with Ythlp, PAP and Rnal4p of CF 1 YTHl 25 Y Ythlp preferentially binds to poly(U) RNA PTA 1 88 Y Previously implicated in pre-tRNA processing PSFl 58 ? PS F2 53 ? otlters?
Notes and references 1. CF II, PF 1 and PAP can be purified as a single complex that has polyadenylation activity (l),
References 1. Preker, P. J., Ohnacker, M., Miiivielle-Sebastia, L., and Keller, W. (1997) Errrbo 116(15), 4727-37 2. Minvielle-Sebastia, L., Winsor, B., Bonneaud, N., and Lacroute, F. (1991) Mol Cell Biol11(6), 3075-87 3. Minvielie-Sebastia, L., Preker, P. J ., and Keller, W, (1994) Science 266(5191), 1702-5 4. Amrani, N.,Minet, M., Wyers, F., Dufour, M. E., Aggerbeck, L. P., and Lacroute, F. (1997) Mol Cell Bid 17(3), 1102-9 5. Minvielle-Sebastia, L., Preker, P. J., Wiederkehr, T., Strahm, Y., and Keller, W. (1997) Proc Nd1 Acad Sei U S A 94(15), 7897-902 6. Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A, B. (1998) Mol Cell2(1), 135-40 7. Kessler, M. M., Henry, M. F., Shen, E., Zhao, J., Gross, S., Silver, P. A., and Moore, C. L. (1997) Geries Deu 11(19), 2545-56 8. Zhao, J., Kessler, M. M., and Moore, C. L. (1997) 1 Bi01 Clrerri 272(16), 10831-8 9. Jenny, A,, Minvielle-Sebastia, L., Preker, P. J,, and Keller, W. (1996) Sciertce 274(5292), 1514-7 10. Chanfreau, G., Noble, S. M., and Guthrie, C. (1996) Scierice 274(5292), 1511-4 11. Russnak, R., Nehrke, K. W., and Platt, T. (1995) Mol Ce11 Biol 15(3), 1689-97 12. Patel, D., and Butler, J. S. (1992) Mol Cd Bi01 12(7), 3297-304 13. Preker, P. J., Lingner, J,, Minvielle-Sebastia, L., and Keller, W. (1995) Cell 81(3), 379-89 14. Barabino, S. M., Hubner, W., Jenny, A., Minvielle-Sebastia, L., and Keller, W. (1997) Cettes Dev 11(13), 1703-16 4.2 3'-processing and the biogenesis of mRNA
Pre-mRNA with a 3'-processing site cm be made exogenously by bacteriophage RNA polymerase in vitro and then added to yeast or mammalian whole-ceil extracts where it will be cieaved and polyadenylated (Butler et al., 1990; Manley et al., 1989). However, by definition, in vivo 3'-processing is coupled to and dependent on transcription since the polymerase does not terminate transcription untii the pre-mRNA has been cleaved at its 3'- processing site (see Termination below). Once again, the CTD and the cap-binding complex play central rotes in coupling transcription to pre-mRNA 3'-cleavage and polyadenylation.
The CTD and 3'-processing A clear relationship between the marnmalian CTD and 3'-processing has been established from in vivo and in vitro biochernical studies. First, in vivo transfection expenments have shown that a hill-length CTD is required for efficient 3'4eavage of reporter transcripts (McCracken et al., 1997). a-amanitin resistant, mouse RNA polymerases with either a truncated CTD (A5;5 heptad repeats) or a wild-type CTD (52 heptad repeats) were transiently transfected into cells dong with a reporter gene with a poly(A) site. Endogenous RNA polymerase activity was inhibited with a-amanitin. RNA was extracted and examined for 3'-cleavage by RNase-protection assays. Reporter transcripts from the ce11 line expressing the truncated RNA polymerase were cleaved 4 to 1O-fold less efficiently than transcnpts made by the wild-type counterpart. Furthemore, RNase-protection probes beyond the wild-type transcript termination site were used to detect the increased presence of run-on transcripts that did not terminate in ceils transfected with the A5 polymerase. Further evidence supporting a role for the CTD was the observation that the CTD binds two of the mammalian 3'-processing factors (McCracken et al., 1997). HeLa extracts were chromatographed sequentially on columns bearing glutathione S-transierase (GST), GST- mutant CTD, GST-wild-type CTD (52 murine heptad repeats), and GST-CTD that was hyperphosphorylated with HeLa ce11 nuclear extract. Eluates were probed with antibodies that recognize two subunits of CstF, three subunits of CPSF and PAP. Al1 of the subunits of CstF and CPSF bound only to the hypo- and hyperphosphorylated GST-CTD columns and not to the GST or GST-mutant CTD control columns in which position 5 of the heptapeptide consensus contained senne to alanine substitutions. Individual subunits of CstF were synthesized by in vitro translation and were then used to show that the p50 subunit of CstF (and the p77 subunit to a lesser extent) was able to bind to the GST-wild- type CTD affimity column. This result has kensupported by the identification of CstF and CPSF as components of the mammalian RNA polymerase 11 holoenzyrne when it is prepared by affinity chrornatography using the elongation factor TFïiS as a ligand (Pan et al., 1997). Since 3'-processing occurs well afier transcription initiation, one might suspect that 3'- processing factors might be recruited by the phosphorylated form of the CTD that is associated with elongating RNAP II. However, there is evidence that these factors may be associated with polymerase when it initiates transcription. The three subunits of CPSF were discovered to be components of an immunopurified form of the TFIID complex (TBP plus TBP associated factors) by microsequence analysis of each of the proteins present in the preparation (Dantonel et al., L997). In a senes of elegant CO-irnmunoprecipitation experiments using purified transcription components and CPSF, it was shown that CPSF is associated with TBP in the pre-initiation complex and as transcription is initiated CPSF becomes associated with the elongating RNA polymerase. Taken together, these No studies show that CPSF may travel with the elongating RNA polymerase complex. This may apply to other processing factors as well, such as CstF. Upon reaching the polyadenylation site, it seems Wrely that these factors dissociate from the polymerase and initiate 3'-processing of the nascent mRNA. The association of 3'- processing factors with components of the initiating holoenzyme may help to ensure that uanscnpts wilI be terminated and processed evea before their synthesis has been initiated.
Zt is uncertain if the CTD plays a similar role in yeast 3'-processing; however. two studies suggest that it may also function in RNA processing. The yeast protein Nrdlp was identified by sequence similarity to the rat proteins rA8 and rA4 (see above and (Yuryev et al., 1996)) as an RNA-binding protein (Steinmetz and Brow, 1998) that is dso capable of interacting with the CTD. Nrdlp was fmt identified in yeast as a factor required to mediate a reduction in pre-MA production from an ACT1-CUPl reporter gene containing a synthetic cis-element engineered into its intron (Steinmetz and Brow, 1996). This cis elernent significantly reduces the accumulation of reporter pre-mRNA and is associated with the appearance of truncated uanscripts which end just downstrearn of the intronic sequence. This defect may be complemented by mutations in the cis element that reduce binding of the RNA sequence to the RRM of Nrdlp. In addition, removal of the CTD- binding domain of Nrd lp is also able to restore transcription from the reporter gene in vivo. Together, these data suggest that the CTD recruits Nrdlp to a cis element in the nascent pre-mRNA and that this somehow mggers transcription termination (Steinmetz and Brow, 1998). The role of Nrdlp in transcriptional elongation andor termination is not yet known; however, another protein that is a subunit of the yeast cleavage and polyadenylation factor 1 (Pcfl Ip) bas amino-terminal sequence sirnilarity with the CTD- binding domain of Nrdlp (Amrani et al., 1997; Steinmetz and Brow, 1996). It will be of interest to determine if Nrdlp is a member of the 3'-processing machinery or if Pcfl Ip is a CTD-associated protein. Such experiments may support coupling between yeast transcription and pre-mRNA processing via the CID. The cap-biruiing cornplex and 3'-processing Efficient 3'deavage of an exogenous pre-mRNA in HeLa extract requires that the substrate be capped (Gilmartin et al., 1988; Hart et al., 1985). In addition, cap analogs inhibit in vitro 3'-processing in much the same way that they inhibit splicing (Hart et al.,
1985). The positive effect of the cap is mediated by the CBC and it has recently been shown that depleting HeLa extract of CBC reduces 3kleavage activity by about 80% (Flaherty et al., 1997). Ln contrast, the cap is not required for polyadenylation of yeast pre-mRNA as demonstrated by at least four studies; however, it is less clear that the cap does not effect
3'-cleavage. First, the kinetics of polyadenylation of uncapped pre-mRNAs appear to be
similar to capped substrate in yeast extract (Butler et al., 1990). Second, temperature- sensitive mutants of the guanylyl-tramferase subunit of the capping enzyme (cegI-63)have no apparent defect in polyadenylation activity at the non-permissive temperature (Fresco
and Buratowski, 1996). Third, a HIS4 transcript synthesized by RNA polymerase 1 in yeast is not capped but is polyadenylated (Lo et al., 1998). Fourth, a 3'-processing site from the GAL7 3'-untranslated region is able to direct polyadenylation of en RNA
polymerase III transcript that is not capped (Liang et al., 1997). Therefore, capped transcripts are not required for 3'-processing as measured by the appearance of polyadenylated product in each of these studies. However, the 3Weavage reaction is not tightl y coupled to the polyadeny lation reaction in yeast extracts (cleavage products are readily detectable in the presence of polyadenylated end-product). One can imagine that the polyadenylation product fkom the rate-limited step may appear unaltered in the presence of less cleavage product. None of the above studies explicitly addressed the requirement of the cap structure for efficient 3'-cïeavage thus this remains a question for future study. 5.1 Termination of transcription and mRNA biogenesis
Termination of RNAP II transcription in eukaryotes requires two cis-acting elements; the polyadenylation site and a downstream element @SE) that is less well defined (Birse et al., 1997). No additional protein factors have been identified that are required for tennination as is the case for Reblp-dependent RNAP I termination (Jeong et al., 1995; Lang et al., 1994) or rho-dependent bacterial RNAP termination (Wu and Platt, 1993). Tennination is coupled to pre-mRNA biogenesis in that it is preceded by transcription of the polyadenylation site and possibly by the 3'-processing event itself. This requirement prevents RNAP JI from prematureiy tenninating transcription before the polyadenylation site habeen transcrïbed. The requirement for the polyadenylation site is weil-documented in both higher eukaryotes (ConneUy and Manley, 1988; Lanoix and Acheson, 1988; Logan et al., 1987; Whitelaw and Proudfoot, 1986) and in yeast (Birse et al., 1997; Russo, 1995; Russo and Sherman, 1989). Ln addition. evidence for the direct requirement of the 3'- processing factors themselves in tennination has recently been obtained in yeast (Birse et al., 1998). Run-on analysis of transcription on a CYCI template indicated that transcription tenninated about 100 nucleotides past the poly(A) site. However, in the absence of a poly(A) site, non-terminated transcription complexes were detected over 600 nucleotides past the normal termination site. This sarne system was used to examine termination under non-permissive conditions in temperature-sensitive mutants of the 3'- cleavage factors RNA II, RNA IS and PCF11 and of the polyadenylation factors PAP, FZPl and YTHI. Termination was abolished in each of the 3'-cleavage factor mutants at the non-permissive temperature, while it was unaffected by any of the factors required only for polyadenylation. It was concluded that efficient 3'sleavage is required for transcription termination. The cleavage and polyadenylation factor (CPSF) and the cleavage-stimulatory factor F (CstF) have been shown to be associated with the CTD of elongating RNA polymerase in higher eukaryotes (Dantonel et al., 1997; McCracken et al., 1997). Presumably , these factors nucleate formation of the 3'-processing complex once they encounter the polyadenylation signal sequence and may provide a mechanism by which 3'-cleavage is coupled to termination; the resulting alteration to the elongating polyrnerase complex may trigger the subsequent tennination of RNA polymerase transcription (Birse et al., 1998; Dye and Proudfoot, 1999; Hirose and Manley, 1998; Osheim et al., 1999). This process may be facilitated by pausing of the polymerase in the downsueam element (Birse et al.,
1997), analogous to the pausing caused by T-rich elements in RNAP 1 and III temiinatm
(Jeong et al., 1995; Matsuzaki et al., 1994). Exactiy how the events of 3'-processing and polymerase pausing combine to terminate transcription is unknown. It bas not been determined whether the yeast 3'-processing factors associate with RNAP iI CTD,thus it is not clear how a sirnilar mechanism might work in yeast or even how mutations in 3'- processing factors could decrease transcription termination. 6.1 RNA export from the nucleus
Following transcription and processing, RNAP II transcripts are exported to the cytoplasm. Both types of RNAP II transcnpts (U snRNAs and mRNAs) are 5'-capped and the presence of this cap dong with the capbinding complex (CBC) has been shown to play an important role in U snRNA transport and possibly an auxiliary role in mRNA export.
U snWA export U snRNAs in higher eukaryotes (with the exception of U6 snRNA which is a RNAP IïI transcript) are exported to the cytoplasm where the& S'-caps are trimethylated and they
are assembled into snRNPs prior to king re-imported into the cytoplasm. The cap was irnplicated in export by early studies showing that microinjection of m7~cap analogs into the nuclei of Xenopus oocytes inhibited U snRNA export (Hamm and Mattaj, 1990).
Furthemore, microinjection of antibodies against the CBC also inhibited U snRNA expon (Izaurralde et al., 1995). Finaily, the CBC has been CO-punfied from Xenopus as a complex with importin-a which binds to capped RNAs. Importin-a shuttles proteins between the cytoplasm and the nucleus and its association with CBC suggests that it may facilitate the export of U snRNAs as well (Gorlich et ai., 1996). Although, it has not been demonstrated that yeast U snRNAs are exported, the yeast CBC (Gcr3p and Mud 13p) are also found in a complex with the yeast equivalent of importin-a (Srplp) (Gorlich et al., 1996). One other protein, CRMl, implicated in U snRNA export (reviewed in (Stutz and Rosbash, 1998)) may also interact with CBC (data not yet published).
mRNA export The S-rn7~~~p~cap is not required for m.Aexpon in the Xenopus oocyte system and its presence has only a two-fold stimulatory effect on the rate of export (Jarmolowski et al., 1994). Furthemore, RNAP II transcripts placed under the control of an RNAP III promoter are exponed sucessfully even though they are not capped (Gunnery and ~Mathews,1995). At best, the role of the yeast CBC proteins in expon is stimulatory rather than mandatory since neither of these proteins is essential to viabiiity and a deletion of the GCR3 gene does not lead to an accumulation of polyadenylated RNA in the nucleus (Gorlich et al., 1996). CIearly, if CBC plays a role in mRNA export, it is either redundant or auxiliary. Export of mRNA in the yeast system most likely requires additiond RNA-binding proteins such as Npl3p (the yeast orthologue of the human hnRNP Al). Npl3p is able to shuttle between the cytoplasm and the nucleus and temperature-sensitive deles accumulate nuclear poly(A)+ RNA (Lee et al., 1996). The precise role that this protein plays in expon is unknown. However, genetic studies have identified two related genes of interest. First, an allele of HRPl was isolated as a suppressor of a temperature-sensitive np13 mutant. Hrplp shuttles between the nucleus and the cytoplasm and has been identified as a component of the 3'-processing machinery (Henry et al., 1996; Kessler et al., 1997). Second, HMTl was identified in a synthetic-lethal screen for Npl3p-interacting functions. Hmtlp is an hnRNP methylase and methylates both Npl3p and Hrp lp, aithough its precise role in export is not clear (Henry and Silver, 1996). Finally, an HMTI deletion allele is synthetically-lethal with a deletion of the largest subunit of CBC (GCR3) (Shen et al., 1998). One could imagine that Npl3p/Hrplp and the CBC-cap compiex are active in different mRNA export pathways and that the synthetic-lethality may be the result of dismpting two cooperative pathways. Altematively, the CBC may be involved directly in the Npl3p export pathway. 7.1 Summary of mRNA biogenesis
The majority of pre-mRNA processing likely occurs concomitantly with transcription. This model draws on data fiom a number of studies with both yeast and higher eukaryotic systems such as fruit aies, mice and humans. Since many of the processes and the proteins involved in mRNA biogenesis are conserved among these organisms, they are often discussed in the literature as though they are directly comparable. For this reason, it is useful to summarize what is known about this model, while making a distinction between which parts of it apply to yeast andor higher eukaryotes. Capping has not been shown explicitly to occur CO-transcriptionallyin yeast; however,
the in vitro, physical interaction between the capping enzymes and the EWAP II CTD in this system make it almost certain that it does (Cho et al., 1998; McCracken et al., 1997). Capping functions to identify nascent mRNAs as RNA polymerase II transcnpts and potenüally tags them as substrates for splicing, 3'-processing, export to the cytoplasm and, finally, translation by ribosomes. To this end, there is evidence from the yeast system that the cap plays at least an auxiliary role in splicing (Colot et al., 1996; Lewis et al., 1996b),
and translation (Gallie, 199 1; iïzuka et al., 1994; Sachs et al., 1997). However, the major function of the cap may simply be to protect mRNAs from 5'-to 3'-exonucleases. Regulated mRNA turnover is essential for the maintenance of a pool of translatable mRNAs in the ce11 and the S'-cap plays an important role in this process; mRNA is degraded rapidly in yeast capping mutants cultured at the non-permissive temperature (Schwer and Shuman, 1996) and mutations in the major cytoplasmic 5'-3' exonuclease (Xmlp) cause the accumulation of decapped rnRNAs with shortened poly(A) tails (Hsu and Stevens, 1993). Increasing evidence indicates that, in higher eukaryotes, the majority of splicing is concomitant with transcription (although post-transcriptional splicing still occurs); co- iocalization and co-irnrnunoprecipitation experiments have demonstrated that factors involved in both processes can be found complexed together on the nuclear matrix (see section 3.2). On the other hand, there is very Iittle evidence that splicing is co- transcriptional in yeast (Elliott and Rosbash, 1996) and there is no evidence to suggest the association of splicing and transcription components in the same complex. Indeed, efficient gene expression in yeast may not require that spiicing be coupled to transcription since less than 4% of all pre-WAs have introns and these introns are comparatively short with strongly-conserved splice sites. However, in supposing that coupling does exist in yeast and in considering how evidence of it might be manifest, it is useîùl to consider what types of interactions one would expect to coupie transcription and splicing in higher eukaryotes. Coupiing factors couid include those that act to juxtapose components of the splicing machinery with the transcription machinery and/or those that facilitate the movement of pre-mRNA substrate from one set of machinery to the next. Under such a scheme, it is possible to imagine that couplhg factors of the first type rnight interact strongly with both transcription and splicing components but have no effect on either of the individual processes when assayed in virro. Altematively, a coupling factor of the second type might interact strongly with RNA but only weakly or temporally with components of the transcription and splicing complexes in order to facilitate coupling so long as stronger interactions of the first type are present to juxtapose the two complexes. This second type of factor might have very little or no effect on the individual processes when assayed in vitro and so detecting such factors will depend on the future development of an in vitro coupied transcription-splicing assay system. Until that time, we are limited to detecting coupling factors of the first type by way of their strong association with both transcription and splicing components. 3'cleavage in both yeast and higher eukqotes is CO-transcriptionalby definition since polyrnerase fails to terminate transcription in the absence of a 3'-processing site (section 5.1). Furthemore, mamrnalian 3'-processing factors interact directly with the CTD of RNA polymerase II (section 4.2). Even though such a physical link has not yet ken demonstrated in yeast, its existence is conceivable given the genetic interaction between 3'- processing and termination in yeast and considering the homology between the CTD- binding domain of Nrdlp and the termination factor Pcfl lp (section 4.2). Finally, it is curious that the CBC has ken implicated in both formation of the early spliceosome complex (in yeast and humans) and later at the furthest end of the pre-mRNA during 3'4eavage (in humans). The action of this complex at two extremes of the nascent rnRNA molecule raises the possibility that the S'-end of the nascent mRNA may not be extruded from the transcription complex as a freely-diffusible element but instead may be tethered by the CBC to a point where it can associate with other splicing/termination factors which in turn could potentially scan passing nascent pre-mRNA for 5'-splice sites or 3'- processing sites. Given this, future analysis of the direct role of CBC in splicingltermination complexes is likely to be useful as a way to understanding how pre- mRNA processing is coupled to transcription. Thesis Summary The fust data chapter of this thesis describes the quantification of the zinc stoichiometry of RNAP ïI and shows that it is 7; consistent with the number of zinc-binding motifs present in the enzyme. During the course of my Master's thesis, 1 constructed a number of mutants in the zinc-binding domain (ZBD) of the largest subunit (Rpo2 1p) of RNAP II and in Chapter II of this thesis 1 continued with the characterization of these mutants. These mutants were assayed with respect to their ability to bind zinc and their effect on the in vitro activity and stabiiity of RNAP II. Mutations in the Rpo21pZBD reduced RNAP II activity in vitro without visibly altering association of subunits required for this activity. In the second data chapter, one of these zinc-binding domain mutants is used in a synthetic-lethai screen in an attempt to identify other components of the transcriptional machinery. Two synthetic-lethal mutations were identified; an ailele of SRBS (a known component of the RNAP IT holoenzyme (Thompson et al., 1993)) and an allele of GCR3 (a factor that had previously been implicated in the expression of glycolytic genes (Uemura and Jigami, 1992). The second data chapter describes experiments aimed at determining if Gcr3p is a component of the holoenzyme and if it is required for efficient promoter-directed transcription in vitro. However, no evidence was found to suggest that Gcr3p is a component of the holoenzyme and its effect on in vitro transcription was marginal and possibly indirect. While this work was in progress, Gcr3p was identified as the largest subunit of the nuclear cap-binding complex and was shown to be important for efficient splicing (Lewis et al., 1996b; Lewis et al., 1996a). 1 was able to show that the allele of GCR3 identified in my synthetic-lethal screen also had an effect on in vitro splicing. 1 hypothesize that this splicing defect, coupled with the transcriptional defect conferred by the zinc-binding domain mutant is sufficient to explain the synthetic-lethality observed between the two. Given that a number of splicing-related factors can interact with the mammalian carboxyl-terminal domain (CTD)of the largest subunit of RNAP II (section 3.2), it seemed plausible that the original genetic screen for suppresors of CTD truncation mutants may have identified components of the splicing machinery (Thompson et al, 1993). One of the mutants identified in this screen was an allele of SRBS. The third data chapter describes an attempt to detennine if SrbSp also plays a role in splicing- Extracts of the mutant SM strain were deficient in splicing activity. However, recombinant WT SrbSp was unable to restore spIicing activity to mutant extracts suggesting that the effect was indirect. In addition, a CïD peptide was unable to inhibit splichg in yeast extract even though it is able to inhibit splicing in HeLa extract (Yuryev et al, 1996). 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Zinc stoichiometry of yeast RNA polyrnerase iI and characterization of mutations in the zinc-binding domain of the largest subunit.
Atornic-absorption spectroscopy was performed at the Banting Institute Trace Elements laboratory by Doug Templeton and Lidija Stuhne-Sekalec and also in Joseph Cole's lab by Kevin Gardner and Matthew Junker. Amho acid analysis was performed at the HSC/Pharrnacia Biotec h center. Abstract
This data chapter demonstrates, by atomic-absorption spectroscopy, that highiy purified preparations of RNA polyrnerase II fiom the yeast Saccharomyces cerevisiae binds 7 zinc ions. This number is in agreement with the number of potential zinc-binding sites present arnong the 12 different subunits of the enzyme. 1 focused on one such candidate zinc- binding motif in the largest subunit (Rpo2lp) of the enzyme to investigate its importance. Mutagenic analysis revealed that altering any one of the six conserved residues within the zinc-binding motif conferred either a lethal or conditional phenotype (Donaldson, 1992). The analysis of these mutants are continued in this chapter. Zinc-blot analysis indicated that mutant forms of the domain had a two-fold reduction in affinity for zinc. Mutations in the zinc-binding domain (ZBD) reduced promoter-specific and non-specific RNA polymerase II activity in ceii-free extracts although protein-blot analysis indicated that the mutant Rpo21p subunit was present in excess with respect to that present in wild-type extract. Purification of one ZBD mutant RNAP II revealed a subunit profile that was WT- like (with the exception of Rpb4p and Rpb7p which were rnissing) even though activity of the core enzyme was only 5% of WT. 1 conclude from these results that mutations in the Rpo2 lp-ZBD can reduce core RNAP II activity without visibly altering the association of any of the subunits required for this activity. Introduction
Zinc is an integral component of both bacterial and eukaryotic DNA-dependent RNA polymerases (RNAPs) and is essential for their function (Coleman, 1983). While the importance of zinc to the function of RNAP is not well understood, a number of studies have suggested that it is generally required to maintain structure that is important for interactions with the 0thpolymerase subunits and/or with DNA/RNA at the active site. Consistent with this hypothesis, bacterid RNAP which is stripped of its two zinc ions
(associated with the p' and fl subunits) fails to re-assemble into the five-subunit active enzyme in the absence of zinc (Solaiman and Wu, 1984). Crosslinking and mapping studies have demonstrated that both the v-subunit zinc-binding motif and a C-terminal domain of the p-subunit make contact with a 7-9 nucleotide, double-stranded DNA region downstrearn of the polymerase active site (Nudler et al., 1996)- These interactions are thought to confer the salt-resistant stability of the DNA-RNA-RNAP ternary complex in its eionpating phase (Nudler et al., 1996). Since the p' and P subunits of E. coli RNAP are homologs of the first and second largest subunits of eukaryotic RNAP (reviewed in (Archmbault and Friesen, 1993; Sawadogo and Sentenac, 1990; Young, 1991)), which also bind zinc (Treich et al., 1991), these results may apply to the eukaryotic RNA polymerases (1, II and III) as well. In support of this, genetic evidence has implicated the zinc-binding motifs of the largest and second-largest subunits of yeast RNAP 1 in mediating a functionid interaction between these two subunits, since phenotypes conferred by mutations in the zinc-binding motif of the largest subunit of yeast RNAP 1 can be suppressed by mutations in the zinc-binding motif of the second-largest subunit (Yano and
Nomura, 199 1). Zinc binds to six of the twelve different subunits of RNA polymerase II (RNAP II) from the yeast Saccharomyces cerevisiae in a zinc-blotting assay (Carles et al., 1991;
Treich et al., 1991). However. the number of zinc ions associated with this enzyme was measured by atomic absorption spectroscopy to be only 1 or 2 (Lattke and Weser, 1976;
Mayalagu et al., 1997). In this chapter, 1 attempt to reconcile this apparent contradiction by measuring the zinc-content of an affiity-purified form of yeast RNAPII. Each of the subunits thought to bind zinc in yeast RNA polymerase contains a zinc- binding motif of the structural type that might coordinate a secondary structure which in turn interacts with protein or with nucleic acids. These types of motifs include at least four potential coordinating amino acids that are predorninantiy cysteines and, to a lesser extent histidines, and these potential ligands occur wiihin a relatively small stretch of amino acids (Berg and Shi, 1996; Vallee and Auld, 1990). One such motif (67~~2~~g~~2~~26~~2~1 *O) occurs in the largest subunit (Rpo2 lp/Rpb 1p) and is conserved in the largest subunits of al1 three polymerases from a variety of eukaryotes (Werner et al., 1992). Part of this motif is also conserved in the E coli P'-subunit (Chatte rji and Guruprasad, 1988). Amino acids R47 to NI 19 of Rpo2 lp containing this motif were expressed as a fusion protein in E. coli and were shown to bind zinc in a zinc-blotting assay (Treich et al., 1991). As a first step to understanding the role of zinc in RNAPII. 1 mutated those residues in the Rpo2lp zinc-binding domain (ZBD) that are both well- conserved and capable of coordinating zinc. This work included detennining the effect of these mutations on cell-viability and was published as part of my Master's thesis (see Figure 2.1 and (Donaldson, 1992)). Six amino acids in the RpoZlp-ZBD have been identified as being both conserved and potentially zinc-coordinating: C67,C70, C77, H80, C 107 and Cl 10 (Werner et al., 1992). Mutations encoding individual substitutions in each of these residues conferred growth phenotypes while mutations in other potentially zinc- coordinating residues in the ZBD did not confer any visible growth defects. This indicates that those ZBD residues that are both highly conserved and capable of coordinating zinc are important to the funftion of RNAP II. This chapter describes the continuation of this work in which I examine the ability of the wild-type and mutant ZBDs to bind zinc in vino. The effects of these mutations on the activity and stabïiity of RNA polymerase II is also addressed here. Figure 2.1: ZBD mutant phenotypes. The substitution-mutations constmcted in the zinc-binding domain of Rpo2lp/Rpblp and the phenotypes conferred when expressed from a low-copy plasmid (Donalcison, 1992). The wild-type sequence of the zinc-binding domain is shown at the top in single-letter atnino acid code. Underlined letters indicate those amino acids which were altered. The position and identity of each mutant (rpo21-27 to rpo21-39) is illustrated beneath. Four multiple mutants (MLM4) were constnicted: Ml(C67S, C70S); M2(C77S, HSOY, H83Y); M3(C105S9 C107S, CI LOS); and M4(C 103S, C105S, C1075, Cl 10s). Growth phenotypes on soiid media at four different temperatures, as determined by plasmid-shuffling experiments, are described: (FOAS) indicates that the strain will not lose the maintenance plasmid at any temperature, (+) indicates growth indistinguishable from the strain transformed with wild-type RP021
(pYFl5 13), (-) indicates an absence of growth and (s) indicates slow growth at the given temperature. The plasmid shuffling assay is described in detail in the Materials and Methods.
aTwo temperature-sensitive mutants previously isolated (Himmelfarb et al., 1987) in this sarne domain. brpcl60 mutants (analogous to those in this study) described in (Werner et al., 1992) and expressed extrachromasomally are shown where (-) indicates a lethal phenotype and
(+) indicates wild-type growth. UT C678 C708 C778 HBOY HB3Y Cl038 Clos8 Cl078 Cl108 Y1 Ma M3 Y4
RPC160 UT rpc160-22k C67A rpcl60- 22 7b C7 01 rpcl 60-220b C77Q rpc160-215~ HBOY rpcl60-11 sb ne3 t rpcl60-23Ob C107A rpci 60-231b CilOA rpcl 60-22sb O7 9D Materials and Methods
Atomic absorption spectroscopy and amino acid analysis RNA polymerase II was prepared using heparin-Sepharose, DEAE-Sephacel, affinity chromatography using 8WG16 monoclonal antibody (Thompson et ai., 1989) and HPLC fractionation essentially as described previously (Edwards et al., 1990) with the following changes. Polymerase was prepared from 450 g of baker's yeast cake stored at -70°C. Protease inhibitors (1 mM PMSF and 2 mM benzamidine) and IO mM DTT were included throughout the purification. The heparin-Sepharose colurnn was washed with five column volumes of buffer A containing 150 mM KCI followed by ten column volumes containing 200 rnM KCI. DE52 resin was used in place of DEAE-Sephacel to remove nucleic acids. Flow-through from the DE52 column was applied directly to 8WG 16-Sepharose beads in 500 mM ammonium sulfate and incubated for four hours with one change of beads. The protein eluted from the 8WG16-Sepharose beads was purified further by HPLC on a Pharmacia Mono-Q column using a gradient of ammonium sulfate from 70 mM to 1M in 50 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 10% glycerof and IO mM DTT (no protease inhibitors were included). RNA polymerase II lacking subunits 4 and 7 (RNAPtIA4/7) was a gift from Aled Edwards that was prepared from yeast strain rpb-4 as described in (Edwards et al., 1990; Edwards et al., 1991).
RNA polymerase II was prepared for atomic absorption spectroscopy and amino acid analysis by dialyzing against three changes of 2 L of buffer D (20 mM HEPES (pH 7.5). 10 m.DTT and 0.5 mM or 10 rnM EDTA) over six hours at 4°C. This was followed by dialysis against three changes of 1 L of metal-free buffer (100 rnM ammonium bicarbonate and 1 mM DTT made metal-free by passage over a Chelex- 100 ion exchange membrane
(Bio-Rad)) over 12 lus at 4°C. SDS-polyacryiamide gel electrophoresis and Coomassie staining revealed that RNA polymerase II subunits represented greater than 99% of the protein present (Figure 2.2). Zinc content was deterrnined by Zeeman graphite fumace atomic absorption spectroscopjj at the Best Institute Trace Elements Laboratory or in Dr. LE. Coleman's lab by Matthew Junker and Kevin Gardner using a Instrumentation Laboratory IL157
spectrophotometer. Zinc content was detemiined by absorption from a standard curve of 1 to 15 pM zinc. Post-column, PITC amino acid analysis of samples was performed at the Best Institute HSCPharmacia Biotech center with 25 nmoles norleucine added to assay recovery of the sample after hydrolysis. The concentration of protein was then calculated from the concentration of arginine, alanine and leucine given the molar ratio of these amino acids to RNAP II or to RNAPIIA4/7 (see (Archambault and Friesen, 1993; Kolodziej et al.,
1990) and references therein). The calculated molecular mass of these polymerases was 572 895 Da or 528 487 Da respectively . His-tagged Rpb9p, Rpb9p-Zn 1(amino acids M 1 to E47) and Rpb9p-Zn2 (amino acids
T55 to S 1 12) were prepared from E.coli by SayHemming (Awrey et al., 1997). Each of the proteins retained three amino acids (GSH)at the N-terminus after removal of the His- tag. Zinc stoichiometry was detennined for these proteins essentially as described above.
Strains and Media Ligation product selections and plasmid amplification were done in the E. coli strain XL 1-Blue (recA - (recAl lac - endAl gyrA96 thi hsdRI 7 supE44 relAl { F' proAl3 IocI Q lac2 AM15 1)) from Stratagene. Fusion proteins were made by the E. coli host TB 1 (ara a(lac proAB) rpsL(phi80 lac2 M15) hsdR) supplied by New England Biolabs. Bacterial media were prepared as described in (Sambrook et al., 1989). Saccharomyces cerevisiae strains used in this study are listed in Table 2.1. Strains
YF1703 and YF215S (constructed by Dave Jansma) are described in (Donaldson, 1992). Plasmids carrying mutant alleles of RP02 1 are listed in Table 2.2. These plasmids were introduced into the RPO2ldeletion strain to create the corresponding strains listed in Table 2.2 (Donaldson, 1992). Yeast media have been previously described: YPD and synthetic Table 2.1: Yeast strains used in this study
W303-la MAT a canl-100 his3-11,lS leu2-3,112 -1-1 ura3-1 ade2-2 W303-lb MAT a canl-100 his3-11,15 Ieu2-3,112 hpl-1 ura3-1 ade2-1 ~~1703~W3031a with rpo21::HIS3 pYF1577(pGALI0-RP021 URA3 CEN ARS) ~~2155~W3û31a with rpo21::HIS3 pYF1513 (RPO21 TRP2 CEN ARS) ~~1733~W303-lb with rpo21::ADE2 pYF1577(pGALlU-RP021 URA3 CEN ARS) notes a. W303-la and W303-lb were obtained from R Rothstein. b. constructed by Dave Jansma Table 2.2: Plasmids used in this study Name - d* tion pYF1513 RP021 TRPl CEN ARS YF2155 pYF1547 rpo21-27 TRPl CEN ARS YF2142 pYF1548 rpo21-28 TRPl CEN ARS YF2143 pYF1549 rpo21-29 TRPZ CEN ARS YF2144 pYFIS50 rpo21-30 TRPZ CEN ARS YF2145 pYF1551 rpo21-32 TRPl CEN ARS YF2146 pYF1552 rpo27-32 TRPZ CEN ARS YF2147 pYF1553 rpo21-33 TRPl CEN ARS YF2148 pYF1554 rpo21-34 TRPl CEN ARS YF2149 (FOAS) pYF1555 rpo21-35 TRPl CEN ARS YF2150 pYF1556 rpo21-36 TRPZ CEN ARS YF2151 pYF1557 rpo21-37 TRPl CEN ARS YF2152 pYF1558 rpo21-38 TRPl CEN ARS YF2153 (FOAS) pYF1559 rpo21-38 TRPl CEN ARS YF2154 (FOAS) pYF1577 pGal-Rpo21 URA3 CEN ARS YF1703 complete (SC) media (Shennan et al., 1986), 5-fluoro-orotic acid (5-FOA) (Boeke et ai.,
1987), no-sulfate synthetic medium (NSM)(Julius et al., 1984). To make low-sulfate synthetic medium (LSM), ammonium sulfate was added to NSM to a concentration of 200
PM-
Plasmid shuffiing assay The plasmid shuffling assay used to assay the phenotypes conferred by zinc-binding domain mutations (see Figure 1) is reproduced here from my M.Sc. thesis for convenience (Donaldson, 1992). Plasmids pYF 1547 to pYF 1559 expressing mutant alleles rpot l-2 7 to rpo21-39 respectively (see Table 2.2) from the endogenous promoter were introduced
(Sherman et al., 1986) into the RP021-gene-disruption yeast strain, YF1703, where the conferred phenotypes were assayed by plasrnid-shuffiing analysis (Boeke et al., 1987); transformants were growa at 23°C or 30°C on SC-glucose medium supplemented with 5- FOA to select for loss of the UM3-maintenance plasmid, pYF1577. Finally, 5-FOAC colonies were streaked to YPD or SC-glucose solid medium lacking histidine and tryptophan at four different temperatures: M°C, 23"C, 30°C and 37°C. These experiments were performed in parallel with pYF15 13 (positive control) and pK39 (negative control) transforrnants of YFl7O3.
Fusion proteins The sequence that encodes amino acids R47 to N119 of Rpo2lp was amplified as a PCR fragment flanked by BamHI and P stI sites using Taq polymerase and the primers
5'-CCCCCGGATCCAGAGCGAAAA'ITGGTC-3' and 5'-CCCCCCTGCAGCTAATTATGTTCATCCAGTAATAG-3and either pYF 15 13, pYF1556, pYF1557 or pYF1559 as template. These fragments were then subcloned in frame to the 3'-end of the coding region for the maltose-binding protein (MBP) in the bacteriai-expression vector pMAL-c2 (New England Biolabs). The resulting fusion- proteins were expressed in TB1 E. coli and purified by affinity chromatography on agarose-maltose beads (New England Biolabs) as described by the manufacturer. The fusion protein names and there respective amino acid changes were as follows: MBP-ZBD (wild-type sequence), MBP-Ml(C67S and C70S), MBP-M2(C77S, HSOY and H83Y), MBP-M4(C lO3S, CIOS, C107S and C 1 10s). Each of the protein preparations was analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970); in each case, a single, Coomassie-blue-stained band was observed corresponding to the expected fusion- protein product of 52 KDa. The ability of these proteins to bind zinc was assayed using an in vitro zinc-blotting assay previously described in (Treich et al., 1991). Negative controls included maltose-binding protein alone (MBP2*; New England Biolabs) and MBP fused to the B-galactosidasea domain (MBP-lad) expressed and purified from strain TB 1 carrying the vector pMALc2.
Preparation of yeast protein exttact Total yeast protein for protein-blot anaiysis and CO-immunoprecipitationswas prepared by harvesting an equivalent of 10 ml of OD600=1.0 yeast culture and washing the pellet in
1 ml cold distilled water. NI subsequent steps were carried out at 4°C. Bufkr A contains 20 mM HEPES (pH7.9), 10 mM EDTA, 1 rnM dithiothreitol (DTT), phosphatase inhibitors [l mM NaN3, 0.4 mM NaV03. 1 mM NaF. 0.1 mglm1 phosvitin], protease inhibitors [5 mM benzamidine, 1 mM PMSF, 0.5 mghl BSA and 10 pg/rni each of aprotinin, antipain, chymostatin, leupeptin, and pepstatin], 10% glycerol and ammonium sulfate. For the purposes of protein-blot analysis, cells were pelleted and resuspended in 2 vol. of buffer A400 (400 rnM ammonium sulfate). One pellet volume of acid-washed glass beads (425-600 microns: Sigma) were added and cells were vortexed 15 X 30 seconds times with 30 seconds on ice in between each round of vortexing. Ce11 debris was removed by centrifugation and the supernatant was collected. Protein concentration was assayed by Bradford anaiysis and supernatants were stored at -70°C. Proiein-Mot analysis
'rcit;d ycrrst proiein (4-40 pg) was electrophoresed on a SDS-polyacrylamide gel (7%
p~~lyricrylrimidcand 0.2% bisacrylamide). The proteins were then electroblotted to lrnmobilon-P membrane (Miliipore) at 0.45 amp constant current for 45 min. in transfer
buffer ( 10 mM CAPS (pH 1 l), 10% methanol). The blot was blocked ovemight in TBST
plus 4% BSA and 1 % non-fat milk at 4OC. Two antibodies were used to detect Rpo2 lp.
The first was the mouse, monoclonal-antibody 8WG16 (kind gift of Richard Burgess
(Thornpson et. al. 1990)) recognizing the hypo-phosphorylated C-terminal domain of Rpo21p (Patturajan et al., 1998). The second was a rabbit polyclonal raised against a fusion of glutathione-S-transferaseand the phosphorylated CïD (gift of Susan McCracken and David Bentley). Rpb2p was detected with primary polyclond-antibody anti-B 150 (kind gift of Andre Sentenac). Secondary goat anti-igG rabbit or goat anti-IgG mouse conjugated to horse-radish peroxidase was from Gibco BRL. Antibodies were diluted in
TBST with 1% BSA. Incubations with antibodies were for 1 hr. at room-temperature each and were followed with 5 X 5 min. washes in TBST at room-temperature. Secondary antibody was detected using ECL reagent as described by the manufacturer (Amersham).
Preparation of transcription extract Whole-ce11 transcription extracts were prepared using a combination of two methods (Schultz et al., 1991; Woontner et al., 1991). Yeast cells were grown at 30°C in 1 L of YPD medium to an O&()() of 1.5-2.0 in log phase. Cells were harvested by centrifugation at 4000 rpm for 15 min. in a Beckman 16-HC rotor. The ce11 pellet was washed once in cold, sterile, distilled water, once in 1.3 volumes of EB (200 mM Tris-acetate (pH &O), 0.39 M ammonium sulphate, 10 mM MgS04, 20% glycerol, 1 mM EDTA, 1 mM Dm) without protease inhibitors and once in 1.3 volumes of EB with protease inhibitors ( 1 rnM PMSF, 10 rnM benzamiàine, 5 pghl leupeptin, 5 pg/rnl pepstatin A) using centrifugation at 4000 rpm for 4 min. in a SS34 Sorvall rotor. The ce11 paste was collected into a 10 ml syringe and extruded into liquid nitrogen. Frozen celis were stored at -70°C. Frozen cells
(2.5-5 g) were placed in a mortar pre-cooled with liquid nitrogen and were broken to a coarse powder with a pestle under liquid nitrogen. After the liquid nitrogen had boiled off, cells were ground to a fine powder using 60 circumferences of the pestle; the celi powder was re-cooled with Liquid nitrogen after every 20 circumferences. The ce11 powder was allowed to thaw partially at 4°C and was then resuspended in 1 ml of EB for every gram of starting ce11 matenal. The extract was spun at 35 Krpm for 2 hr in a 70.1 Ti rotor at 4°C.
The protein in the supernatant was precipitated by adding ammonium sulfate to 0.34 g/ml of supernatant. Precipitated protein was pelleted by centrifugation at 25 Krpm for 15 min. in a 70.1 Ti rotor at 4 OC. The pellet was dissolved in TDB (20 mM HEPES pH 7.5, 20% glycerol, 10 mM MgS04, 10 mM EGTA, 5 m.DIT, 1 mM PMSF) using 60 pl for every gram of starting yeast cells. The extract was dialyzed for 12 hrs against 1 L of TDB with two changes of buffer. Conductivity of a 1 in 100 dilution was measured to ensure that it was equivalent to that of TDB. Extract (typically 20-40 ~&1as determined by Bradford assay) was frozen in smail aliquots in Iiquid nitrogen and stored at -70°C.
RNA polymerase activity assay Assays for RNA polymerase activity were perfonned essentially as described by (Ruet et al., 1978) with modifications. Reactions were performed in a 30 pl volume. The reaction mix (25 pl) contained 50 mM Tns-acetate (pH 8.0), 3 mM MnC12,600 ph4 ATP, GTP and CTP, 20 pM UTP, 2.5 pCi &~P]-UTP (3000 Ci/mmol; Mandel), 50 ng/@ native, calf-thymus DNA, 50 n&1 heat-denatured, calf-thymus DNA, 5 rnM DTT, and 4 units RNasin (Promega). Al1 reactions were performed in either the presence or absence of
10 pg/ml a-arnanitin (Sigma). Reactions were started by adding 5 jU of extract (typically containing 20 pg of protein) in TDB buffer and allowed to proceed at 23°C for 5-30 min. Reactions were stopped by adding 10 pl stop bmer (80 mM EDTA, 2% SDS, and 2 pg/@ proteinase K) and incubation at 37°C for 20 min. The reaction mix was spotted (25 pl) to a 2.4 cm diameter DE81 filter (Whatman) which was washed three times in P-buffer (350 mM Na2HP04, 10 mM sodium pyrophosphate) for 5 min. each, followed by 3 times in water for 2 min. each and finally by a rinse in ethanol. Filters were dried, added to scintillation vials containing 7 ml Ecoscint and counted in a LKB 1217 liquid scintillation counter. Al1 reactions were performed in tripkate. Error between identical reactions was generally less than 5%. Counts per minute in no-DNA control reactions represented less than 5% of counts in DNA-containhg reactions.
In vitro, promoter-specific transcription assays Promoter-specific transcription assays were performed essentially as described (Lue et al., 199 1) with modifications. Reactions were in a total volume of 30 pl. A reaction mix of 23 pl consisted of 50 mM HEPES (pH 7.6), 90 m.potassium glutamate, 0.75% poly- ethylene glycol (MW 4000), 10% glycerol, 5 rnM EGTA, 10 mM magnesium acetate, 2.5 mM DTT, 4 units of RNasin (Promega), 10 units RNase T 1 (BRL),0.15 pg creatine kinase (Sigma), 30 mM creatine phosphate, 20 pM UTP, 400 ph4 CTP and ATP, 0.2 rnM 3'-O-methyl-GTP, 200 ng pBR322 competitor DNA, 300 ng circular, template DNA: pGAL4CG-(see ((Lue et al., 1989)); aka pYF1643), and 5 pCi [a-32~1-UTP(3000 CUmmol; Mandel). The reaction was initiated by adding 7 pl of extract (typically containing 80 pg of protein) in TDB to the reaction mix. Reactions were incubated at 23°C for 1 hr unless otherwise stated. Reactions were stopped by adding 10 pl stop mix (200 mM NaCl, 80 mM EDTA, 2% SDS, 2 mg/d proteinase K (BRL)) and incubating the mixture at 37°C for 20 min. RNA was precipitated from the reactions by adding 4 pl sodium acetate, 20 pg E. coli tRNA (Sigma) and 300 pl EtOH followed by mixing and microcentrifugation for 10 min. The pellet was washed with 70% EtOH, dried by speed- vacuum and was finally resuspended in 17 pl sdH2O and 23 wl of loading buffer (90% formamide, IX TBE buffer, 2.5 mghl bromophenol blue and 2.5 mghl xylene cyanol). II- 17
Samples were heated to 65°C for 5 min. before loading 20 pl on a pre-run, 0.8 mm, 6%
poIyacrylarnide/7 M magel. Electrophoresis was at 350 v for 2 hrs. The gel was dried at 80°C for 30 min. and then exposed to a Molecular Dynamics phosphor-imager screen for 6 to 12 hrs.
Cell labelling and immunoprecipitation of RNAP II For each imrnunoprecipitation, 10 ml of cells grown to an ODmof 0.5 were labelled wi th [3%]methionine as described in (Kolodziej et al., 1990; Kolodziej and Young. L 99 1) at their permissive or non-permissive temperature for 2 hours. Preparation of the extracts and immunoprecipitations were done at 4°C. Yeast protein extract was prepared by glass- bead lysis as described under "Preparation of Yeast Protein Extract". The extract was diluted with an equal volume of buffer A (without glycerol) containing 100 mM ammonium sulphate, 10% non-fat dry milk, 1 mghl BSA and 2% Triton X-100. The extract was pre- cleared by incubation with protein A-sepharose CL-4B beads (Sigma) for 30 min. Beads were removed by centrifugation and 1 pg of 8WG16 monoclonal antibody (Thompson et al., 1989) was added. After a 60 min. incubation, protein A-sepharose was added and incubation was continued for another 60 min. Beads were collected by centrifugation and washed twice with buffer B400 (20 mM HEPES (pH 7.9)- 10 rnM EDTA, 400 mM ammonium sulfate) and once with buffer B50 (20 mM HEPES (pH 7.9), 10 mM EDTA, 50 rnM ammonium sulfate). Beads were then mixed with a standard gel-loading buffer (Laernmli, 1970) and heated to 65°C for 10 min. before electrophoresing on a SDS- polyacrylamide gel (12.5% polyacrylamide and 0.3% bisacrylamide). Each of the immunoprecipitated subunits were identified by matching them to their CO-migratingbands in a purified RNAP II preparation detected by Coomassie-blue staining. The gel was dried onto Whatman papa and exposed to BIOMAX-MR film (Kodak) or to a Molecular Dynamics phosphor-imaging screen. Results Zinc Stoichiometry of RNA polymerase II It has been shown that six of the yeast RNAP II subunits are capable of binding zinc in
an in vitro zinc-blotting assay (Carles et al., 199 1; Treich et al., 199 1). However, it has been estimated from atomic absorption analysis that only one or two zinc ions are bound by
this enzyme (Lattke and Weser, 1976; Mayalagu et al., 1997) . In an attempt to resolve this apparent contradiction, I prepared an affrnity-purified form of RNAP II (see Methods and Figure 2.2) and determined its zinc content. The highiy purified nature of this enzyme allowed me to determine protein concentration by amino-acid analysis. Zinc content was deterrnined by atomic-absorption spectroscopy for three independent preparations. The molar ratio of zinc to polymerase was found to be 7.26M.27 following dialysis of the
enzyme against 10 mM EDTA at 4OC foiiowed by rnetal-free bufTer (see Methods and Table 2.3). These samptes bound comparable amounts of zinc when dialyzed against only 0.5 mM EDTA. In addition, 1 used a preparation of RNAP lIA4/7, which lacks subunits Rpb4p and Rpb7p. Neither of these two subunits contains a zinc-binding motif, nor are they able to bind zinc in an in vitro zinc-blotting assay. Furthemore, this polymerase is indistinguishable from RNAP II in promoter-independent transcription assays. The molar ratio of zinc to RNAPM4/7 was calculated to be 7.0.22(Table 2.3). 1 conclude that the nurnber of zinc ions bound by RNAP II is one more than the number of subunits that are
abIe to bind zinc in an in vitro zinc-blotting assay. Rpb9p has two potential zinc-binding motifs and therefore it could account for at least two of the zinc ions bound to RNAP II. 1 measured the zinc bound independently by Rpb9p and to the two halves of Rpb9p (Rpb9p-Zn1 and Rpb9p-Zn2; each containing one zinc-motif) produced in E. di. 1 found that these proteins bound 2.3kû.25,0.7M.08 and 0.7&û.07 zinc ions, respectively (see Methods and Table 2.3). Figure 2.2: RNA polymerase II preparation used for atomic-absorption
spectroscopy. Affinity-purified RNA polymerase II ( 15 pg) was electrophoresed on a SDS-polyacrylamide (15%) gel. This polymerase and two other identical preparations were used for quantification of zinc stoichiometry. Migration of subunits are indicated on the left-hand side and pre-stained, molecular mass standards (kDa) are shown on the right.
The preparations were judged to be greater than 99%pure by Coomassie-blue staining; only one other minor band appeared below Rpb2p at about 70 kDa. Rpbl Op - . -6 Rpbl2p Table 2.3: Zinc-stoichiometry of RNA polymerase II
RNAP U dialvzed anainst metai-free buffer with 10 mM EDTA
2 0.98H.01 7.4 7.58 3 1.77M.01 12.5 7.08 Average 7.260.27
RNAP II dialyzed against metal-free buffer with 0.5 mM EDTA
P@p~~me~L"_:-~~~~~~~6BBzE2iw-, -+#%. w-m-+&-&.T-V7?:% -- zz7- ~~-~"~.-?x.-a. ..* +?!>$ L:0; 2 L?Le">< * -2L - e *.-sJ---- * -.- .+--3 1 1.lW.01 9.95 9.05
RNAP ii A4/7 dialvzed anainst metal-free buffer with no EDTA
Rpb9p and derivatives dialvzed against metal-hee buffer with no EDTA In conclusion, approximately 7 zinc ions are bound by RNAPII, two of which are accounted for by Rpb9p alone. This value fails within the expected range of potential zhc- binding ability of RNAP II based on the number of zinc-binding motifs present and the stoichiometry of each of the subunits. It seems likely, from the data presented above, that each of these motifs may in fact bind zinc, and that the zinc content of RNAP II was originally underestimated (see Discussion).
In vitro zinc-binding analysis. Since substitutions of amino acids that are both highly conserved and capable of coordinating zinc in the RpoSlp-ZBD confer growth phenotypes, it is possible that these same substitutions might reduce the ability of this domain to bind zinc. The Rpo2lp-ZBD
(amino acids N47 to N119) was produced in E. coli as a fusion to the maltose-binding protein (MBP). This fusion was previously shown to bind zinc in an in vitro assay (Treich et al., 1991). In addition to the wild-type fusion (MBP-ZBD), three mutant versions were produced that carried amino acid substitutions C67S and 00s(MBP-Ml) or C77S, H80Y and H83Y (MBP-M2) or C103S, C105S, C107S and Cl 10s (MBP-M4). These fusion proteins were separated by electrophoresis on an SDS-polyacrylamide gel, blotted to PVDF membrane, and then were probed with 65~nradioisotope (Figure 2.3).
Wild-type MBP-ZBD fusion protein was able to bind zinc (Figure 3B lane 4). This binding activity was attributed to the ZBD portion of the fusion protein since neither MBP2* (lane 2) nor MBP-lada (lane 3) bound detectable levels of zinc in this assay. Zinc-binding was quantified and normalized to protein by staining the membrane with amido-black. This reveaied that the zinc bound by mutant fusion proteins MBP-Ml, MBP-
M2 and MBP-M4 was reduced to 40%- 60% and 45% respectively compared to that bound by the wild-type MBP-ZBD.1 conclude that mutations in the zinc-binding domain reduce its affinity for zinc. 1 further hypothesize that a deficiency in zinc-binding underlies the Figure 2.3: Zinc-blot analysis of the zinc-bindiag domain (ZBD) of RpoZlp in its wild-type and mutant forms. Proteins were electrophoresed on 10% SDS-PAGE and then electroblotted to PVDF membrane. The membrane was probed with 65~n~12and then were stained with amido black (A) following autoradiography (B).Lane contents: M: molecular weight markers (29,43 and 68 kDa) from BRL;MBP2*: maltose- binding protein (MBP) (42.7 kDa); MBP-IacZa: MBP fused to the B-galactosidase-a domain (52 Da); MBP-ZBD: MBP fused to amino acids R47 to Ml9 of Rpo2lp (ZBD)(S1 -2 kDa); MBP-ZBD fusion protein with amino acid substitutions C67S and C70S is MBP-Ml; with amino acid substitutions C77S, HBOY and H83Y is MBP-M2; with amino acid substitutions C103S, C105, Cl07 and Cl 10s is MBP-M4. B
% 'zinc-binding' decreased RNAP II activity (see below), and the growth phenotypes conferred by these sarne mutations.
Effect of ZBD mutations on RpoZlp steady state-levels and RNAP II stability Figure 2.4 shows that (1) the growth rate of the Cl 10s substitution mutant begins to slow two hours after shift to the non-permissive temperature and (2) that the ce11 mass ceases to increase by 24 hours afier shift. In addition, 1 assayed the abiiity of these cells to grow on solid medium at various times after shift. Ce11 viability began to decrease after 5 hours of shifi to the non-permissive temperature (data not shown). This pattern was sirnilar for both mutants Ml and M2 although mutant M 1 took longer to cease dividing and shifted cells could recover when transferred to solid medium at the permissive temperature even after 30 hours of shift. In general, mutations in the zinc-binding domain confer a slow shut-off phenotype when shifted to the non-permissive temperature. Such a phenotype is consistent with a weak defect in Rpo2lp stability, in core RNAP II stability/assembly and/or in RNAP II activity. Some amino acid substitutions in the Rpo2 lpZBD could confer temperature-sensitive phenotypes. It has also been demonstrated that underproduction of the largest subunit of RNAP II can confer temperature-sensitive growth phenotypes (Archambault et al., 1996). Therefore, 1 hypothesized that the stability of the largest subunit rnight be reduced by substitutions in the zinc-binding domain. In order to address this possibility, 1 chose to examine levels of Rpo2lp in the YF2150 (Cl 10s) strain before and after shifting to 37°C. The C 1 10s allele confers the most severe temperature-sensitive phenotype of the zinc- binding domain mutants. The data shown in Figure 2.4C suggest that a stability defect is unlikely; extracts prepared from mutant and WT cultures grown at the permissive temperature showed that Cl 10s had increased levels of Rpo2lp even though this mutant grows more slowly than the WT main at 23'C. Furthemore this increase persisted at least Figure 2.4: Effect of a CllOS substitution mutation on Rpo2lp steady- state levels as determlneà by protein-blot analysis. A. slow shut-off phenotype at 37°C of strain YF2 150 carrying the Cl 10s substitution allele of RP021. The WTyeast strain (YF2155) and mutant were inoculated from 23°C log-phase cultures to YPD media pre-warmed to 37°C. Culture growth in YPD medium was monitored by absorbance at 600 nm for 28 hours. Growth of the WT cuïture is shown only for the fmt six hours after the shift to 37°C. B. The growth rates of those cultures from which protein extracts were prepared for anaiysis of Rpo2lp and Rpb2p protein levels in panel C. Equivalent amounts of extract (20 pg) were electrophoresed in each lane on a SDS-polyacrylamide gel (7%), blotted and probed with 8WG16 and anti-Rpb2p. Signal was detected using secondary antibodies conjugated to horse-radish peroxidase and ECL (Enhanced Cherniluminescence). Images were scanned and quantitated using NM Image software.
Rpo21p and Rpb2p signal were quantified with respect to that present in 20 pg of WT extract (lane 1), 10 pg and 5 pg of extract (not shown). O 10 20 30
Time after shift to 37°C (hrsl
O 5 10 15
Time (hm) 2 hours after temperature shift (data not shown). Even at 6 hours after shift the level of Rpo2 1p in C 1 10s was comparable to that of WT grown at the permissive temperature
(compare lanes 1 and 4). Mutants Ml, M2 and H80Y also showed increased Rpo2lp levels at the permissive temperature (30°C) which persisted for at least 2 hours after shift to
the non-permissive temperature (data not shown). Steady-state levefs of both Rpo2 lp and Rpb2p did decrease in the C 1 IOS substitution mutants at 12 and 24 hours (lanes 5-6); most likely, this reflected an overall decrease in RNAP II levels as the cells began to die. 1 considered the possibility that increased levels of Rpo2 1p as detected by 8WG 16 antibody
might indicate increased levels of hypo-phosphorylated Rpo2 1p (Patturajan et al., 1998) rather than overall increased levels of the subunit since the monoclonai antibody 8WG 16 preferentially recognizes the hypo-phosphorylated form of the C-terminal domain of RpoSlp. 1 probed protein blots with a polyclonal antibody raised against the phosphorylated C-terminal domain of Rpo2lp. and confimed that levels in mutants were indeed increased with respect to that found in WT extracts (data not shown). 1 conclude
from this that the temperature sensitive defects conferred by mutations in the zinc-binding domain are not due to an overaii decrease in the steady-state levels of Rpo2 lp. In order to confirm that the ZBD mutants conferred an RNAP II defect, 1 prepared whole-ce11 extracts from WT and mutant strains (Ml, M2 and H8OY) grown at the permissive temperature of 30°C . Each of the mutant extracts were reduced by about two- fold for RNA polymerase II activity in assays that used heat-denanired calf-thymus DNA as template (Figure 2.5 A). This defect was specific to RNAP XI activity since RNA polymerase 1 plus polymerase III activity in these same mutant extracts was at least 90% of that present in WT extracts (Figure 2.5 A). 1 also assayed these extracts in a promoter- specific assay (Lue and Kornberg, 1987). Mutants were able to produce full-length, accurately initiated transcripts (Figure 2.5 B). However, again there was a sirnilar decrease in overall activity indicatïng that the mutations iikely affect only the promoter-independent Figure 2.5: Transcription activity of ZBD mutant extracts. Panel A: Equivalent amounts of celi-free extract (20 pg) from WT (YF2 155) and each of the mutants M 1 (YF21S 1), M2 (YF2 152) and HSOY (YF2 145) were assayed for RNA
polymerase activity in a transcription assay using calf-thymus DNA as template for 20 minutes at 23°C. The amount of extract and the time of reaction used were both shown to be non-saturating. Totai RNA polymerase activity or RNA polymerase 1 plus JII activity was measured, respectively, in the absence or presence of a-arnanitin. RNA polymerase II
activity was cdculated as the difference between the two. Activity was expressed as a percentage of that found in WT extracts. Each of the bars represents the average of three
trials with an error of less than 5%. The expriment was carried out with two sets of
ex tracts . Panel B: Equivalent amounts of ce11 fkee extract (80 pg) made from the sarne strains were also assayed for promoter-specific activity at 23OC using template DNA that has a G-less cassette. Transcnpts initiated from this promoter do not incorporate guanosine and so they
are not degraded in the presence of RNase Tl. Transcnpts (labelled using a[32~]-UTP) from two separate start sites of 375 and 350 nt. were detected by a phosphor screen (Molecular Dynamics) following denaturing pol yacrylamide gel electrop horesis. Numbers below each lane represent promoter-specific transcription product relative to WT. Quantification of the data was with Image-Quant software (Molecular Dynarnics). Panel C: Protein-blot analysis of RpoZLp levels in WT and mutant extracts. The whole- ce11 extracts used above were adjusted to equivalent protein concentrations (as determined by Bradford assays). Increasing volumes of each extract were electrophoresed on a SDS- polyacrylamide gel (7%), blotted and probed with 8WG16. Rpo2lp signal was detected by ECL. Images were scanned and quantified using NIH Image software. Numbers below lanes indicate signal strength with respect to signal from an equivalent volume of WT extract. relative FüUAP act ivity (%) on denatured cal-thym- DNA .ki!, ~f HSA pi~lymcwand have no spccific effect on initiation. No increase in the
,kfcLr u .L\ enw i~hincrcasrng tcmpcnture indicating that the growth phenotype is likely a
:r\i:ii 01 inirra.~drnailtviiy to reduced RNA polymerase II activity at higher temperatures
:.if hcr i!un IO ;l tcnlpcnturc-sensitive gene product.
3lutri:icms in rhc zinc-binding domain of the RNAP III largest subunit were reported
pn-\ WU^- to alter the association of smaller subunits with the core enzyme (Werner et al.,
1 992 ). I hrprhesized that mutations in the analogous RNAP II ZBD aiso alter association of othcr subunits with the core enzyme. This possibility was examined by detennining whcther ail of the RNAP iI subunits could be CO-imrnunoprecipitatedby the largest subunit containing a ZBD mutation. Whole-ceIl extracts were prepared from three mutant and one wiid-type culture that had been labelled with [3%]-methionine at 23OC or 37°C. Rpo2lp was immunoprecipitated from each dong with its associated subunits which were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography (Figure 2.6). None of the WT RNAP II bands observed were completely rnissing in the mutants at either the permissive or non-permissive conditions. Bands corresponding to subunits
Rpb 1, 2 and 3 were observed at both the permissive and non-permissive temperatures. Bands corresponding to subunits RpbS, 6 and 8 were also detected but were much fainter especially for mutants Ml and M2 at the non-permissive temperantre since less Rpo2 lp was recovered from these samples. 1 cannot rule out the possibility that association of some of the smaller subunits is affected since these were either not resolved well or were too poorly labelled in order to be detected. The bands were quantified and nomalized first to the number of methionines present in each subunit and then to the Rpb2p band present in each lane (Table 2.4). Quantifjing changes in core enzyme stoichiometry was difficult due to lower signal to background ratio in mutants. The most obvious and reproducible difference was the excess of Rpo2lp with respect to the other subunits in Cl 10s at both temperatures. Rpo2 lp was also in excess of the other subunits Figure 2.6: Analysis of stoichiometry of RNAP II immunoprecipitated from wild-type and mutant ce11 extracts. Yeast cultures carrying either wild-type RPOSILUPBI or substitution alleles Ml (C67S), M2 (C77S,HSOY and H83Y) or C 1 10s were Iabelled at the permissive temperature of 23°C or at the non-permissive temperature of 37°C with [%]-methionine. Rpo2 lp and associated proteins were immunoprecipitated from ce11 extracts using the monoclonal antibody 8WG16. hunoprecipitated proteins were electrop horesed on a SDS-polyacry larnide gel, dried and autoradiographed or exposed to a phosphor screen. Immunoprecipitated RNAP II subunits were identifîed by co- migrating, Coomassie-stained bands in a purified preparation of RNAP II that was elecuophoresed aiongside (marked on rïght). Signal corresponding to each of the subunits was normaiized to the number of methionines present in the subunit and then expressed as the number of subunits present in the immunoprecipitation relative to Rpb2p (defined as 1 in Table 2.4 below). A control immunoprecipitation with anti-retinoblastoma antibody (lane 1) indicated that no significant background bands CO-rnigratedwith RNAP LI subunits except for Rpb4p and Rpb7p (quantification for these subunits is not shown). Quantification was performed with Image-Quant software (Molecular Dynarnics) using rectangles encompassing each band and correcting for local background.
- - - Table 2.4: Summary of quantification of Figure 2.6 expressed as number oi subunits ~resentrelative to RD~~Din each immunomeci~itation
(by about 2-fold) in M 1 and M2 when compared to the WT. This can be explained by the excess Rpo2 lp present in these mutants. Although other minor changes were noticeable
between mutant and WT polymerase profiles, 1 was unable to find any stability defect that was significant and reproducible using this method. 1 cannot rule out the possibility that some finer stability defect exists in the mutants that cannot be detected here. In order to further investigate the possibility that ZBD mutations alter the stability of the
core RNAP II, 1 prepared enzyme from one of the mutants (M 1; C67S, C70S) in exactly
the same manner described for preparing WT RNAP II for the zinc-stoichiomeuy determinations. Figure 2.7 shows that each of the RNAP II subunits is present in the M 1 preparation with the exception of Rpb4p and Rpb7p. Each of the subunits was quantified frorn a scan of a Coomassie-stained gel using NIH Image software. Subunits Rpb2p, Rpb3p, RpbSp, Rpb8p, and RpbBp+I lp were present in stoichiometries (relative to Rpo2 lp) identical to those for WT RNAP II. Rpb6p stains poorly with Coomassie and could not be quantified; however, it was found to be present by silver staining (Figure 2.7
B). Both Rpb9p and 1 lp were detected and resolved by silver staining. A band corresponding to RpblOp and 12p was present in both the mutant and WT enzymes, but the two subunits were not resolved. Despite the presence of each of these subunits, the Ml RNAP 11 was 20-fold less active in transcription assays using denatured calf-thymus DNA as template (Fig 2.7 C). This decrease in activity cannot be explained by the absence of Rpb4p and Rpb7p since these subunits are only required for promoter-directed transcriptional activity (Edwards et al., 1991) (assuming that Rpb4p and Rpb7p are not essential in the presence of a mutant ZBD). These observations demonstrate that mutations in the ZBD can decrease basal activity of the enzyme without observably altering the presence of any subunits required for this function. 1 cannot conclude that the Ml ZBD mutation is causative of the absence of Rpb4p and Rpb7p because the Ml RNAP II was prepared from cells grown to late log phase and the WT polymerase from yeast cake (stationary phase). This might account for the absence of Rpb4/7 from the Ml Figure 2.7: Comparison of subunit profile and activity in WT and mutant Ml RNAP II preparations
A. Mutant Ml (C67S, C70S) RNA polymerase II was prepared fiom 300 g of yeast strain
YF2 15 1 grown to late log phase. Preparation of the polymerase was exactly as descnbed for WT RNAP II from 500 g of yeast cake (see Materials and Methods). Mutant Ml RNAP II was electrophoresed on an SDS-polyacrylamide gel (14%) (10 pg in lane 1) and compareci to 16, 12, 8 and 4 J.L~of WT RNAP iI (lanes 2-5) by Coomassie staining.
Mutant M 1 poiymerase was prepared from 300g of RNAP II subunits are indicated to the left of panel A. Rpblp is the same as Rpo21p. B. Silver stain detection of smder subunits present in 2.5 pg each of Ml RNAP II (lane 6) and of WT RNAP II (lane 7) resolved on an SDS-polyacrylarnide gel (14%). RNAP II subunits are indicated to the left of panel B. C. Mutant Ml and WT RNAP II (130-330ng) were assayed for RNA polymerase activity in a transcription assay using calf-thymus DNA as template for 20 minutes at 23°C. Each of the points represents the average of three trials with an enor of less than 5%. -WT RNAP II
100 200 300 400 RNAP Il (ng) preparation (at least in part) because the stoichiometry of RpWpincreases with respect to other RNAP XI subunits in stationary phase (Choder and Young, 1993). Having said this, other WT and mutant RNAP II preparations made from late log phase ceiis (not €rom yeast cake) using the method described here do contain Rpb4p and Rpb7p (Sally Hemming and Steve Orlicky; persona1 communication), therefore the absence of Rpb4/7p from the Ml mutant preparation is unexpected. It is estimated that the stoichiometry of Rpb4p in RNAP II is 0.2 in log phase cells and that this increases to 1 in stationary phase ceils (Choder and Young, 1993). Discussion
The evidence presented here supports results of zinc-blot analysis showing that zinc is bound by six of the twelve different RNAP II subunits. One of the subunits (Rpb9p) binds two equivalents of zinc. This is the fust study showing a correlation between the number of zinc ions bound by RNAP 11 in vitro and the number of zinc-binding motifs present in the core enzyme. The three largest subunits and the three smallest subunits of RNAP II are able to bind zinc in a zinc-blot assay (Carles et al., 1991; Treich et al., 1991). Given that Rpb9p is able to bind two zinc ions (this study) there is a potential for RNAP II to bind at least seven zinc ions (see Table 2.5). The actuai number of zinc ions bound may be more or less than this depending on the stoichiometry of Rpbgp, Rpb 1 Op and Rpbl2p (see notes to Table 2.5). Indeed, I found that RNAP II was able to bind 7.26M.27 zinc ions. In addition, 1 found that 7.W.22 zinc ions were associated with RNAPIIA4/7. These results are more consistent with the number of RNAP II subunits able to bind zinc in an in vitro blotting assay than with the earlier, independent estimations of 1 or 2 zinc ions (Lattke and Weser,
1976; Mayalagu et ai., 1997). The large difference between this result and those published previously may be due to a combination of factors including the method of RNAP II purification and preparation for atomic absorption spectroscopy. In this study, RNAP II was dialyzed against either 0.5 rnM EDTA or 10 rnM EDTA at 4Twith no apparent difference in the number of zinc ions bound. This higher concentration of EDTA was shown previously to remove half of the zinc associated with the enzyme afier a four hour dialysis at room temperature (Mayalagu et al., 1997). This raises the possibility that binding of zinc to RNAP II rnay be less stable at higher temperatures. My zinc determination differs in one other important respect; 1 was able to determine protein concentration from amino acid anaiysis. This is essential to obtaining an accurate, absolute Table 2.5: RNAP II zinc-bindine cavacitv RPB subunit zinc-bindhg motif potential total notes stoichiometrv zincs bound
9 1-2 7~~2~~18~~2~322 2-4 &hi ~~cx~cx~~cx~c~~ IO 1 ~CX~CX~~CC~~ 1 1 jtk 12 1 ~~cx~cx~~cx~c~~1 1 I,k Total 7-9
Notes and references to Table 25.
(a) also known as Rpo2lp (see (1)) (b) stoichiometry from (2) (4 (3) (4 (4) (e) stoichiometry from (5) (f) non-canonical motif may explain weak-binding of subunit in zinc-blot assay (6) (8) (7) (h) stoichiometry of Rpb9p was estimated as 2 (2) before it was resolved from a co-migrating subunit Rpbllp (8) and so may have been over-estimated (i) zinc-binding determined in this study (j) also known as ABClOb and RPBlOb (sec (9,lO)) (k) stoichiometry of RpblOp was determined before it was resolved from Rpbl2p. The stoichiometry of both is assumed here to be 1 for the purposes of calculation. (1) also known as ABClOa, RPBlOa and RPClO (see (10,ll)) (m) the zinc stoichiometry for each of the subunits (except Rpb9) is assumed to be at least 1 from the ability of these subunits to bind zinc iri vitro (see (6,lO)) Notes and references to Table 2.5 (continued)
Allison, L. A., Moyle, M., Shales, M., and Ingles, C. J. (1985) Cdl42(2), 599-610 Kolodziej, P. A., Woychik, N., Liao, S. M., and Young, R. A. (1990) Molecrrlnr G. Cellirlnr Biolqy 10(5), 1915-20 Sweetser, D., Nonet, M., and Young, R. A. (1987) Proceeditigs of the NRtionnl Acndertiy of Scimces of the Utiited States of A tilericn 84(5), 1192-6 Kolodziej, P., and Young, R. A. (1989) Molecrrlnr & Cellrrlar Biology 9(12), 5387-94 Svetlov, V., Nolan, K., and Burgess, R. R. (1998) 1 Bi01 CIiern 273(18), 10827-30 Treich, I., Riva, M., and Sentenac, A. (1991) 1 Bi01 Clteni 266(32), 21971-6 Woychik, N. A., Lane, W. S., and Young, R. A. (1991) joirrnal of Biological Ckeniistry 266(28), 19053-5 Woychik, N. A., McKune, K., Lane, W. S., and Young, R. A. (1993) Ge~ieExyressioti 3(1), 77-82 Woychik, N. A., and Young, R. A. (1990) \oitninl of Biologicnl Chemistry 265(29), 17816-9 Carles, Ce,Treich, I., Bouet, F., Riva, M., and Sentenac, A, (1991) j Bi01 Clreni 266(35), 24092-6 Treich, I., Carles, C., Riva, M., and Sentenac, A. (1992) Cette Expr 2(1), 31-7 value which cannot be detennined reliably fiom relative proteinconcentration methods such as those used in previous measurements. Other eukaryotic RNA polymerase II enzymes have been reported to bind various numbers of zinc ions (reviewed in (Coleman, 1983) ranging from 2.2 for Euglena gracilis (Falchuk et al., 1976) to six for wheat germ RNAP KI (Petranyi et al., 1977)). These differences may reflect a different number of zinc ions bound by each polymerase. Altematively, each polymerase may bind a similar number of zinc ions that are not always retained depending on the method used to purify the polymerase and prepare it for atomic absorption spectroscopy . The zinc-binding domain of the largest subunit of RNAP II was chosen for further analysis. Changes to potential zinc-coordinating amino acids of the Rpo2lp-ZBD that are conserved among dl three eukaryotic RNAPs conferred, for the most part, heat and cold- sensitive growth defects (see Figure 2.1 and (Donaldson, 1992)). In addition, RNAP II
activity is specifically decreased in extracts made from these mutants (Figure 2.5 A, B). These observations demonstrate that the conserved arnino acids of the ZBD are important to
the function of RNA polymerase II as was shown for the analogous domain in RNAP III (Werner et al., 1992). Mutations in the Z8D reduce the ability of this domain to bind zinc in an in vitro zinc-blotting assay (Figure 2.3) and 1 hypothesize that this loss in affinity for zinc underlies the phenotypic and activity defects observed for these mutants. In considering how mutations in the ZBD rnight affect RNAP II activity 1 addressed three possibilities. The ZBD mutations rnight cause a defect in the level of the RpoZlp subunit, in core RNAP II stabilitylassembly ancilor in RNAP JI core activity itself. The possibility that the ZBD mutations simply caused a decrease in Rpo2 lp levels was consistent with the conferred phenotype. It has been shown that yeast carrying a sole copy of RP021 under the control of a pLEU2 promoter produce ten-fold less Rpo2 Ip when grown in the presence of leucine (repressing conditions) (Archarnbault et al., 1996). Under these conditions, cells displayed both a temperature-sensitive growth defect and an inositol auxotrophy similar to that observed for the zinc-binding domain mutants. However, 1 observed that steady-state levels of RNAP II in the Cl10s ZBD mutant were WT-like even six hours after shifi to the non-permissive temperature when mutant ce11 growth had already started to slow down (Figure 2.4). 1 conclude from these observations that a loss of Rpo2lp is not causative of the growth defect obsewed for the C 1 10s mutant. in addition, 1 found that RpoZlp levels were actually increased with respect to WT levels in mutant extracts prepared at the permissive temperature even though RNAP II activity in these extracts was reduced (Figure 2.5). This suggests that an Rpo2 lp feedback mechanism may be at work in the case of the ZBD mutants since RpoZlp levels are normally not in excess of the other subunits in WT cells (Kolodziej and Young, 1991). The ZBD mutations rnight trigger the overproduction of Rpo2 lp in one of two ways; they rnight decrease efficient assembly of Rpo2lp into the core polymerase or they might reduce the activity of core RNAP 11. In either case, a decrease in functional RNAP Il would occur that would not be corrected for by the feedback mechanism but would instead lead to an excess of the subunit. Evidence of an Rpo21p feedback mechanism was demonsuated previously by showing that artificially repressing levels of Rpo2lp led to enhanced protein expression of a P-galactosidase reporter gene under the control of the RP021 promoter
(Archarnbault et al., 1996).
Next, 1 considered the possibility that ZBD mutations might alter the association of other subunits in the core RNA polymerase. This seemed to be a likely possibility based upon three previous studies. First, bacterial RNAP which is stripped of two zinc ions (associated with the p' and p subunits) fails to re-assemble into the five-subunit active enzyme in the absence of zinc (Solaiman and Wu, 1984). Second, genetic evidence has implicated the zinc-binding motifs of the fmt- and second-largest subunits of yeast RNAP 1 in mediating a functional interaction between these two subunits, since phenotypes conferred by mutations in the zinc-binding motif of the largest subunit of yeast RNAP 1 can be suppressed by mutations in the zinc-binding motif of the second-largest subunit (Yano and Nomura, 1991). Third, and most relevant, a mutation in the zinc-binding domain of the largest subunit of yeast RNAP III (analogous to the domain studied here) resuits in an unstable enzyme and correlates with the dissociation of three of its srnaller subunits (Werner et al., 1993). For these reasons, I examined the association of core subunits with
Rpo2lp in three of the ZBD mutants by irnmunoprecipitating polymerase from [35~]- methionine labelled extracts grown at 23°C. 1 found no significant alterations in the stoichiometry of subunits associated with Rpo2lp using this method (Figure 2.6). In addition, 1 found that a preparation of mutant Ml RNAP II possessed subunits present in WT stoichiometric ratios with the exception of Rpb4p and Rpb7p. This mutant Ml polymerase was reduced in transcriptional activity to 5% of WT RNAP II levels. These observations demonstrate that mutations in the ZBD can decrease basal activity of the enzyme without observably aitering the presence of any subunits required for this function. The association of three subunits (Rpc82p, Rpc34p and Rpc3 lp) with RNAP iII are decreased in the presence of a mutation in the ZBD of Rpo3 lp (Werner et al., 1993). These three subunits interact with one another to form an RNAP iII subcomplex (Werner et al., 1993) and mutations in either RPC34 or RPC31 decrease in vitro promoter-directed transcription by RNAP but not non-specific transcription activity from poly[d(A-T)) templates (Brun et ai., 1997; Thuillier et al., 1995). This in vitro phenotype is reminiscent of RNAP II lacking the Rpb4pRpb7p subcomplex; the absence of these subunits decreases RNAP II promoter-directed transcription but not non-specific transcription (Edwards et al.,
199 1 ). This raises the intriguing possibility that the Rpc82p/Rpc34p/Rpc3 1 p subcomplex is a functionai homologue of Rpb4p/Rpb7p and that each subcomplex interacts with the ZBD in the largest subunit of the respective RNA polymerase. Deterrnining whether the Rpb4/7p subcomplex functionaiiy interacts with the Rpo2lp-ZBD may be resolved by further experiments. At this point, the absence of RpbWp from the Ml mutant is probably not relevant to the loss of transcription activity for this mutant since these subunits are not required for non-specific transcription from denatured calf-thymus DNA. This assumes that RpMp and Rpb7p are not required for transcription activity in the presence of a mutant RpoZlp-ZBD. Thus, at this time, 1 am hypothesizing that the hinction of the Rpo2lpZBD
is related to the basal activity of core RNAP II. Studies with E. coli RNA polymerase aiso point to a role for zinc that is more closely
related to the activity of core polymerase. A double cysteine to serine substitution mutant in the p' subunit of E. diRNAP (analogous to the Ml mutant investigated here) was shown to be inactive in vivo and the mutant polymerase to be less processive than the wild-type in vitro (Nudler et id., 1996). In addition, cross-linking studies identified a domain that included the p' zinc-binding motif dong wi& conserved region 1 of the p subunit as being associated with a 7-9 nucleotide stretch of dsDNA downstream of the RNAP active center (Nudler et al., 1996). It was shown further that these interactions are responsible for conferring the salt-resistance of the elongating temary complex. The punfied E. coli mutant RNAP produces a nurnber of early-tenninated transcripts in an in vitro transcription assay. An extract made from the corresponding M 1 mutant in this study showed no signs of early-terminated transcripts in a promoter-specific assay (Figure 2.58 and data not shown). This difference between the E. coli and yeast mutant may indicate differences between the prokaryotic and eukaryotic enzymes or may relate to differences in the function of other proteins present in the yeast extract that was used for the promoter-specific assays. In any case, my results are not inconsistent with a roIe for the yeast ZBD in maintaining processivity of the polymerase since activity in both non-specific and promoter-specific assays was reduced for the ZBD mutants (Figure 2.5). The results from the E. coli RNAP study suggest that the corresponding domains in yeast Rpo2lp and Rpb2p also interact with dsDNA near the active center. However, using gel-shift analysis, 1 did not detect a non-specific DNA binding ability for the MBP-ZBD (amino acids R47-N119) fusion protein (not shown). In addition, 1 did not detect a pairwise interaction between the Rpo2 1p-ZBD (amino acids M 1-L 14 1) and region 1 (amino acids LI058 to F1224: (Sweetser et al., 1987)) of Rpb2p using the yeast two-hybrid system (Fields and Song, 1989). If the proposed interaction does indeed occur, it may reqüire additional regions of Rpo2lp andor the presence of dsDNA. It is also likely that other components of the yeast core polymerase or holoenzyme may also be required if an interaction between the Rpo2lp-ZBD and region 1 of Rpb2p is to be replicated in vitro since the interaction between the equivaient domains in E. coli RNA polymerase is dependent upon the polymerase undergoing a transition to the elongation state. References
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Young, R. A. (1991). RNA polymerase II. Annual Review of Biochemistry 60,689-7 15. Chapter III A synthetic-lethal screen for factors that interact functionally with R PO2 1
Some of the initiai screening for synthetic-lethai mutants described in this chapter was performed by Scott Houliston under my supervision. Sequencing of gcr3-100 was done at the HSC-Phamacia Biotechnology center. Abstract This chapter describes the use the rpo2l-30 (H80Y)zinc-binding domain mutant in a synthetic-lethal screen designed to identify additional components of the transcriptional machinery. Two synthetic-lethds were identified: an allele of SRB5 (a known component of the RNAP II holoenzyme) and an allele of GCR3 (a factor that had previously been implicated in the expression of glycolytic genes). I examined the possibility that Gcr3p was also a component of the holoenzyrne and that it was required for efficient promoter- directed transcription in vitro. No evidence was found to suggest that Gcr3p is physically associated with the RNA polymerase II holoenzyme; the effect of a gcr3 mutant dele on in vitro transcription was marginal and possibly indirect. Gcr3p was identified (by another group) as the largest subunit of the nuclear cap-binding complex and was shown to be important for efficient splicing. The allele of GCR3 identified in this synthetic-lethai screen also affected in vitro splicing. 1 hypothesize that this defect, coupled with the transcriptional defect conferred by the zinc-binding domain mutation, is sufficient to explain the synthetic-lethality observed between the two. Introduction
1 set out to use the zinc-binding domain mutants, which 1 previously isolated, in a genetic screen to identify other components of the transcriptional machinery that functionally interact with RP021 in general or with the zinc-binàing domain specifically. Comrnonly used genetic screens include suppressor screens and synthetic-lethal screens. Two mutations are said to be syntheticdy lethal with one another if, by themselves, they confer non-lethal phenotypes, but, when present together in the same cell, prevent growth. Obviously, such inviable combinations cannot survive by themselves so in practice a wild- type version of one of the genes is supplied on a 'maintenance' plasmid and the synthetic- lethal combination is identified by virtue of the fact that the maintenance plasmid cannot be lost. Synthetic-Iethai mutations rnay be in the sarne gene (intragenic) or in two separate genes (extragenic). Extragenic synthetic-lethals may arise in two genes that are involved in the same pathway. These mutations by themselves may confer in vitro defects (in biochemical assays of the relevant pathway activity) that can distinguish them from other non-informative mutations in unrelated pathways. For this reason non-lethal mutations cm be used to screen for secondary synthetic-lethal mutants that represent new factors in a given pathway. Syntheticaily lethal mutants may be Iethal together because they alter two gene products that act sequentially in different steps of a single pathway or that act together at a single step of a pathway. For exarnple, one could imagine that a mutation in a component of the RNAP II holoenzyme that reduces the efficiency of transcription initiation might be tolerated in an otherwise wild-type cell; however, a second mutation that reduces the rate of transcription elongation rnight sufficiently lower the overall efficiency of gene expression so that viability becomes impossible. Synthetic-lethai screens have been successful in many studies; for example, in the identification of many additionai components of the nuclear pore complex (Fabre and Hurt, 1997) and of the spliceosome (Xu et al., 1998). 1 chose to use one of the zinc-binding domain mutants as a starting mutation in a synthetic-lethal screen to identie additional components of the transcriptional machinery. When this plan was conceived, it was just becoming clear in the scientifk literature that RNA polymerase was part of a larger complex of proteins (the holoenzyme) that was responsible for regulated transcription in vivo. Many of the proteins associated with this complex were unidentified and others were not essential for ce11 viability. Given this, a synthetic-lethal screen was a feasible method to identiQ other components of the holoenzyme. In addition, in the event that some of the identified synthetic-lethal alleles turned out to be specific to the zinc-binding domain mutants, then some insight into the function of this domain could be gained. The rpo2I-30
(HSOY) mutant was chosen as a starting mutation because it conferred a suong no-growth phenotype at 37°C and had no visible growtb defect on solid media at 30°C (Chapter 2). In this chapter, 1 describe the identification of five synthetic-lethal mutations in a screen that started with rpo21-30. Two of these mutations are shown to be extragenic to rpo2 1-30 and both of these mutations by themselves (Le., in the presence of wild-type RP021) confer an in vitro transcription defect. The first mutation is shown to be an allele of SRBS (a known component of the RNAP Il holoenzyme; see Chapter 1, Section 1. l), which dernonstrates that the rationale of the synthetic-lethal screen is sound. The second mutation lies in the GCR3 gene. Previous studies with GCR3 have suggested that this gene plays a role in transcription (see Discussion). These observations from previous studies, together with my identification of GCR3 in the synthetic-lethal screen, were consistent with the idea that this protein could be another subunit of the RNAP 11 holoenzyme. I describe work in this chapter that attempts to determine if Gcr3p is associated with RNAP II or if it functions in transcription. No conclusive evidence was found for either role. However, as this work was proceeding, Gcr3p was shown to be the yeast onhologue of the largest subunit of human cap-binding protein (Gorlich et al., 1996). The cap-binding complex has been implicated in splicing (Chapter 1, Section 3.2), 3'4eavage of nascent transcripts in the mammalian system (section 4.2) and in export of U snRNAs transcribed by RNAP II (section 6.1). The gcr3 allele isolated in this screen was found to confer an in vitro s plicing defect . I hypothesize, therefore, that the transcription defect conferred by rpo21-30 coupled with this splicing defect are responsible for the observed synthetic- iethality observed between RP02I and GCR3. Materials and Methods
Media and yeast methods Synthetic-complete media (SC), nch media (YPD)and SC medium supplemented with
5-FOA are described in Chapter 2. Red colour development was facilitated on solid medium by doubling the usual dextrose concentration to 4% (wlv) in YPD agar and adenine was limited to 20 mg/l in synthetic-complete agar medium. Construction of diploid strains and tetrad dissections are as described (Sherman et ai., 1986; Treco and Winston, 1997). Gap-recombination (or plasmid gap-repair) is as described (LundbIad and Zhou, 1997).
Construction of strains for synthetic-lethal screen Strain YF1733, constructed by D. Jansma (Archambault et ai., 1996), was used to introduce mutant alleles of RPO21 into the chromosome; the chromosomal copy of RP021 in this strain has been disrupted by insertion of the ADE2 gene between the BstE2 (-722) and SpeI (+622) sites of RP021.. For the synthetic-lethai screen, strains carrying either the rpo21-30 or WT RPO2l alleles on the chromosome were made by transforming yeast strain YF1733 with plasmid pYF1550 or pYF15 13 cut with HindiïI. Transformants able to grow on glucose medium were screened for an Ade- (pinkcolored) phenotype. Finaily, transformants were selected in which the pYF1577 maintenance plasmid was lost on 5- FOA medium, indicating those transfomants (YF2066 and YF2070) in which the chromosomal rpo21::ADEt allele had been replaced by the allele carried on the transforming plasmid DNA (rpo21-30 and RPO2l) respectively. At least two transfomants of each dele were tested for growth phenotypes at 23"C, 30°C and 37OC on YPD medium. Table 3.1: Yeast strains used in this study (part 1 of 2) -- . Strain relevant genotype
MATa cntr 1-100 liis3-11,15 leir2-3,112 tryl-1 itra3-1 ade2-1 ssdl-d (aka YF554) MATa cnrr2-100 his3-11,15 leit2-3,112 trpl-1 rtrn3-1 nde2-1 ndc3::HisC ssdl-d CAL psit (aka YF2035 from Fatima Cvrckova; derivative of W303-la) W303-lb with rpo21::ADE2 pYF1577(pCAL10-RP021 URA3 CEN ARS) MATa rp21-30(H80Y) caril-100 his3-11J5 leit2-3,112 trpl-1 iira3-1 nM-1 ssdl-d (derived from YF1733) MATa RF021 cartl-100 liis3-11,l5 lei12-3,112 trpl-1 irra3-1 ade2-1 ssdl-d (derived from YF1733) MATa rpo21-30(H8OY) cntil-100 liis3-11 ,15 leit2-3,112 trpl-1 irra3-1 de2-1 ssdl-d nde3::HisG (derived from YFZO66) hlATa RP021 cari 1-100 his3-11 ,15 leu?-3, 112 trpl-1 itrn3-1 ade2-1 ssdl-d ade3::HisC (derived from YF2070) MA Ta rpo2 1-3O(H8OY) cnnl-100 liis3-11 ,15 Iert2-3, 112 trpl-1 iirn3-7 nde2-1 ssdl-d nde3::NisC (pYF1465(RPO2 1 URA3 A DE3 2p)) derived from YF2074 MATa R PO21 cm11-1 00 his3-11 ,15 leir2-3, 112 trpl-3 rrra3-1 ade2-1 ssdl -d ode3::HisG (pYP1465(RP021 UAA3 ADE3 2pM)) derived from YF2075 srb5-100 UV-induced mutant of YF2079 (aka P47) gcr3-100 UV-induced mutant of YF2079 (aka 1'88) YF2105 transformed with pYF1466 (FOAr) YF2111 transformed with pYF1466 (FOAr) MA Tala K2346 x YF2O74 MA Ta RP021 canl-100 kis3-11 ,15 leii2-3, 112 ttyl-1 irra3-1 nde2-1 ssdl-d ade3::HisC YEp13 (LEU2)(spore product of Y F2O93) Table 3.1: Yeast strains used in this study (part 2 of 2) Strain relevant genotype YF2097 MATa y021-30(H80Y) cad-100 hid-11 ,15 krr2-3, 112 trpl-1 itra3-1 de2-1 ssdl-d ade3::HisC (spore product of YFZO93) YF2107 MATala YF2105 x YF2097 YF2113 MATala YF2111 x YF2097 YF2330 MATa KP021 carrl-100 liis3-11,15 leir2-3,122 f ty3-1 irrn3-1 ade2-1 ssdl-d gcr3::HIS3 (derived from YF2070) Y F2341 MATala YF2105 x YF2094 YF2342 MATala YF2111 x YF2094 YF2344 MAT ? RP021 srb5-100 nde2-2 adc3::HisC (spore product of YF2341; aka Tl-1) Y F2347 MAT 7 RP021 srb5-100 nde2-1 ade3::HisC (spore product of YF2341; aka T7-4) YF2349 MAT ? RP021 gcr3-IO0 ade2-1 ade3::HisG (spore product of YF2342; aka T14-2) YF2350 MAT ? RP022 gcr3-100 ade2-1 nde3::HisG (spore product of YF2342; aka T14-4) Y F2380 W3031a with rpo21::HIS3 pYFl409(RP021 URA3 CEN ARS) YF2382 W3031a with rpo2l::HIS3 pYF1513(RP021 TRPl CEN ARS) Y F2384 W3031a with rpo21::HlS3 pYF1562(RP021 TR Pl CEN ARS) Y HU3003-2B MATO Agcr3::URA3 le112-3,lI2 irrn3-52 his6 (see (Uemura et al, 1992); aka YF2220) Table 3.2: Plasmids used in this study
Name description source (see references in textl
RP021 URA3 CEN ARS (aka pJS121/pJH121) Howard Himmelfarb (Jim Friesen) A DE3;;HisG::URA3::HisC;::ADE3 (ADE3 disruption cassette) Fatima Crckova (Kim Nasmyth) rpo21-30 TRPl CEN ARS Chapter 2 RP021 URA3 ADE3 2pM RP021 from pYF1337 in pTSV31A RP021 LEU2 AUE3 2pM RPO2l from pYP1337 in pTSV30A SRB5 (original library Sau3A subclone) URA3 CEN ARS library subclone RP021 TRPl CEN ARS (aka pDJ2O) Dave Jansma (JimFriesen) rpo21-RI 2 URA3 Xiao Hua (Jim Friesen) RF021 TRPl CEN ARS (aka pJA483 and pYF1406) Jacques Archarnbault Uim Friesen) sitl-4 TRPl CEN ARS Dave Jansma (Jim Friesen) SRB5 LEU2 CEN ARS PCR fragment from pYF1494 in pRS315 pCd-Rp21 URA3 CEN ARS Jacques Archambault Uim Friesen) GCR3 (original library Sau3A subclones) URA3 CEN ARS library subclone gcr3 (BarnHI-end-filled) URA3 CEN ARS BamHI end-fil1 of pYF1618 GCR3 (Ch1 fragment) URA3 CEN ARS ClaI fragment of pYF1618 in YCpW GCR3 (Sad site follows ATG) URA3 CEN ARS PCR-mediated mutagensis of pYF1693 CCR3 (N-termial HA tas) URA3 CEN ARS oligo insertion in pYF1697 CCR3 (N-termial HA tag) LEU2 2pM ClaI fragment from pYF1707 in YEp351 CCR3 (N-termial Hisl2HA tag) URA3 CEN ARS oligo insertion into pYF1707 Yeast strains YF2074 and YF2075 were constructed by transfonning strains YF2066
and YF2070 with plasmid pYF1455 (aka c 18 16; obtained from Fatima Cvrckova and Kim Nasmyth (Alani et al., 1987; Cvrckova and Nasmyth, 1993)) that had ken digested with SphI. The SphI fragment carries an ADE3::HisG:: URA3::HisG::ADE3 disruption cassette. Single-step gene conversion of the ADE3 locus with this fragment yielded Ura+, white transfonnants (ade2, ade3). These transformants were then spread on solid medium supplemented with 5-FOA to select for spontaneous loss (by intrachromosomal recombination at the HisG locus) of the UR.3rnarker, leaving a dismpted ADE3 gene (ade3::HisG) on the chromosome. Strains YF2074 and YF2075 were transformed with pYF1465 (RP021, ADE3, URA3, 2-ph4) to yield strains YF2079 and YF208O. Both of these strains are red on medium lacking uracil and are able to produce red and white sectors at 30°C on rich
medium (YPD). YF2079 was used to isolate mutations that are synthetically lethal with rpo2 1-30. The same steps described above were used to create straïns that could be used to isolate mutations that are syntheticaiiy lethal with rpo21-27,-28 and -29 (see YF2076-
YF2078).
Construction of plasmids for synthetic-lethal screen The Sun-BamHI fragment from pYF1361 containing the 7 Kb HindIIl chromosomal fragment of RPOZl was subcloned to the San-BamHI cloning site of pYFi337 (aka pTSV31A constmcted by Mike Tibbits in John Pnngle's lab; 2-pM, ADE3, URA3) or pYF1336 (aka pTSV30A constmcted by Mike Tibbits in John Prïngle's lab; 2-pM, ADE3. LEUZ) to yield plasrnids pYFl465 and 1466. Either of these plasmids when introduced into yeast strain YF2074 confers red and white sectoring at 30°C on YPD and allows the strain to grow at 37°C. The pYF1361 source of the 7 Kb Hindm fragment used to make the maintenance plasmids pYF1465 and 1466 was also parental to the Hindm fragment carrying rpo2 1-30 that was used to replace the chromosornai RP021 locus in YF1733. Therefore, the RPO2l sequence inctuding upstream and downstream sequences between the Hi& sites (fiom -1583 to +5410 where +1 is the A in the initiating ATG of RP02I ORF) is essentiaiiy identical between the chromosomal copy and the maintenance plasmid copy in the YF2079 strain used to isolate synthetic-lethals (see Figure 3.2). Al1 DNA manipulations were performed essentiaiiy as descnbed in (Struhi. 1987). WT RP021 constmcts
niree plasmids canyïng WT aiieles of RP021 were assayed for their ability to rescue the synthetic-lethality of strain YI2133 and 2134. Since each of these constmcts differs in their ability to rescue the sectoring phenotype, I have included as much detail as is known about their construction. pYF1513 (constructed by Dave Jansma (aka pDJ20); see also Chapter 2); the chromosomd RPP021 Hindm fragment (nt - 1585 to +543 1 where the A of the ATG is +1) was subcloned from pYF1361 to the Hindm site of pK39 (whose EcoRI site had previously been destroyed by end-filling with Klenow). The source of RP021 for pYF 1361 was from pJAY36 (constructed by Jacques Archambault). 1 was unable to trace the source of RPO21 for pJAY36. pFL39 is a TRPL CEN. ARS plasmid (Bonneaud et ai., 1991). The promoter of MO21 in pYF1513 is closest to the Cid site of the polylinlcer in pFL39 (see Figure 3.1). pYFl562 (constructed by Jacques Archambault; aka pJA483); the chromosomai EcoRI-
Hindm fragment of RP021 (nt -3 14 to nt 543 1 where +l is the A of the initiating ATG) was subcloned to the EcoRI-Hindm sites of pFL39 (Bonneaud et al., 1991). 1 was unable to trace the source of RP021 for pJA483 (see Figure 3.1). pYF1409 (aka pJS 12 1; equivdent to pJH 12 1 (constructed by Howard Himmelfarb
(Ingles et al., 1984)) but with XhoI site of pAPS2 destroyed by end-filling): the chromosomal EcoRI fragment of RPOZ1 (nt -3 14 to nt +856 1) subcloned to the EcoRI site of pAPS2 (pINT2) (Percival and Segall, 1986) (see Figure 3.1). Figure 3.1: WT RP021 plasmids. Numbering is from the Saccharomyces cerevisiae Genome Database (November, 1998). ORFs on either side of RP021 include
BPLl (biotin:protein ligase) which extends from nt 7524 to nt 545 1 and YDLi39C
(unknown function) which extends from nt -1557 to nt -8 14. Details on the construction of each of these plasmids are in the Methods section. H=HfndIIl, E=EcoRI
III- 15
Construction of plasmids containing GCRJ The numbering for GCIUcontaining fragments is based on +L as the A of the initiating ATG. The OWextends from +l to nt 2905 (Gm.The ORF is intempted by an intron that extends from nt +26 GTATG) to nt +347 (TAd). The correct N-terminal amino acid sequence then is MFNRKRRGDFDE (Uemura et al., 1996). The 4.2 Kb ClaI fragment containing GCR3 (from nt -576 to nt +3637) was subcloned from pYF 16 18 to the ClaI site of YCpSO (URA3, CEN, ARS) to create pYF1693.
pYF1618 was cut with BarnHI (unique site in the GCR3 ORF at nt +871), end-filled with Klenow fragment of DNA polymerase 1 and religated to create plasmid pYF1672.
A unique SacI site was introduced after the initiating ATG of GCR3 using PCR. The frst PCR reaction amplified sequence upstrearn of the ATG using oligonucleotides: ID059 (5'-d(G-296AGAGAGA~CAGAAATTGAAATGC-272)-3) and IDOoO (~'-~(GGGGAGCTCC~AT~AAATATA~AAAATGC-~O)-~'. The SacI site is underlined and subscripts refer to the GCR3 nucleotide number of the preceding letter. The second PCR reaction amplified sequence downstream of the ATG using oligonucleotides: ID06 i (5'-d(WGA~~4TTAATAGAAAAAGAAGA~)-)and
ID062 (5'-d(C 13 1TGTCACACCGTACTAACïG 1 1 2)-3')
The first PCR product was digested with SnaBI (nt -201) and Sad. The second PCR product was digested with Sad and XbaI (nt +62). These PCR products were then ligated to one another, digested again with SacI and XbaI and then ligated with pYF 1693 that had previously ken digested with SnaBI and XbaI and treated with alkaline phosphatase. The resulting vector (pYF1697) contained a unique SacI site following the initiating ATG of GCR3 such that the predicted N-terminal amino acid sequence was changed from
MFNRKK ... to MELFNRKK ....The plasmid pYF1697 was able to restore sectoring to yeast YF2 134 just as well as pYF1693. A heme-agglutinin epitope tag (amino acid sequence YPYDVPDYA; (Kolodziej and Young, 1991)) was introduced into the unique Sacl site of pYFl697 using oiigonucleotides:
ID06 3 (5'-d(CtacccatacaacatcccagactacgctCAGCT)-3 and
ID064 (3'-D(TCGAGatgggtatgctacauqqtctgatgcgaG)-5'
A unique AarII site is underlined. The bold letters indicate a nucleotide change that destroys one of the two SacI sites. The resulting plasmid (pYFl707) had a single oligonucleotide pair inserted in the correct orientation at the SacI site such that the predicted N-terminal amino acid sequence was MELYPYDWDYAQL... (where the HA-epitope is underlined). Sequence analysis indicated no other mutations between the SnaBI (nt-201) site and the XbaI site (42). pYF1707 was able to restore sectoring to yeast strain YF2 134 and protein-blot analysis of extracts made from transformants detected a new 100 KDa band using anti-HA antibody 12CA5. The 4.2 Kb ClaI HA-GCR3 fragment from pYF1707 was end-filled with Klenow and subcloned to the SmaI site of YEp35 1 (LEUZ. 2-pkf)to create pYF1720. This plasmid was able to restore the slow growth phenotype of YF2220 (YHU3003-SB) and protein- blot analysis of extracts made from these transformants revealed a new 10KDa band using anti-HA antibody.
A 6XHis-tag was introduced into the Sad site of pYF 1707 using oligonucleotides
ID067 ( 5 ' -d (CcatcaccatcaccatcacGAGCT)-3 'and
ID068 (3'-d(TCGAGgtagtggtagtggtagtgC)-5').
The Sac1 site sequence is capitalized. Sequence analysis of the resulting plasmid
(pYF 177 1) reveaied that two tags had been inserted to give a predicted N-terminal amino acid sequence of MELHHHHHHELHHHHHHELYPYDVPDYAQL (where the HA- epitope is underlined). A gcr3::HIS3 disruption strain was made using PCR-rnediated, one-step gene disruption (described in (Baudin et al., 1993) and (Lundblad et al., 1997)). Oligonucleotides ID069 and ID070 were used to ampli@ the HIS3 marker gene from plasmid pRS303 with GCR3-homologous sequence on either end. ID069 5'-d(C-39GCGTITGGGGCTACAA~GCA~AAATATA~MG-
1ctgtgcggtatttcacaccg)-3' ID070 5'-d(G2g47CGGAGTGATAACGAATGTAGTCCATCCTCCGAATC'ITT2~aga
ttgtactgagagtgcac)-3' Upper-case sequence corresponds to GCR3 and subscripts refer to the position number of the preceding nucleotide. Lower-case letters correspond to sequence on either side of the marker genes in each of the pRS30x senes of plasmids. The 1.3 Kb PCR product was introduced into strain YF2070. Transformants were selected for on SC-His and then screened on nch medium (YPD)for a slow-growth phenotype at 37°C. These slow- growing transformants were screened for the presence of a new 1.5 Kb PCR product that could be amplified between pnmers DO59 and ID070.(see above). Three independent
GCR3-disruption strains were generated in this way ;YF2330-YF23 32.
UV mutagenesis Yeast strain YF2079 was grown in SC-Ura medium to mid-log phase (107 cellslrnl). Cells were concentrated 10-fold by centrifugation at 3000 rpm for 3 min. in a Sorvall SS- 34 rotor and resuspension of the ce11 pellet in SC-Ura medium. Twenty ml of concentrated cells were placed in a Petri dish (8.5 cm diameter) and were exposed to W light (254 nm) in a Spectrolinker XL-150 for some tirne (45 seconds) chat had been empincally shown to cause 90% mortality. Cells were spread on YPD agar medium at a density of about 500 cells per plate and were incubated at 30°C for 4-5 days in the dark before being screened for red (non-sectoring) colonies. Cloning of P47 and P88.
The yeast genomic library CEN BANK A was obtained from the ATCC (#37415). This library carries a partial Sau3AI digest of yeast genomic DNA subcloned into the BarnHI site of the plasmid vector YCpSO (URA3, CEN4, ARSI) (Thrash et al., 1985).
Transformation of yeast was as described in (Hill et al., 199 1). Preparation of yeast DNA for rescue of the library plasmid was modified from (Davis et al., 1980). Briefly, IO ml of yeast culture grown to an O&j()0<3.0 was harvested by centrifugation. The pellet was washed in distilled water and then resuspended in 0.5 ml of 1M sorbitol, 100 mM EDTA. Zymolase LOO-T from Arrhrobacter luteus (20 pl of 10 mg/ml from ICN 100,000 Wg) was added and the cells were incubated at 37°C for 60 minutes. Cells were pelieted by centrifugation and resuspended in 500 pl of 50 mM Tris-
Cl (pH 7.4), 20 mM EDTA plus 50 pl of 10% (w/v) of SDS and incubated at 65°C for 30 minutes. Potassium acetate (200 pl of a 5 M solution) was added followed by incubation on ice for 60 min. with occasional vortexing. The solution was centrifuged in a micro- centrifuge at maximal speed for 10 min. at 4°C. The supernatant was collected and extracted once with chloroform. DNA was precipitated by rnixing with 750 pl of isopropanol at -20°C for at least 15 min. The DNA was pelleted by centrifugation for 10 min. at maximal speed in a micro-centrifuge at 4°C. The pellet was air-dried and then dissolved in 25 pl of 1X TE. Two to five pl of this DNA solution was used for transformation into comptent E. coli. Sequence from either end of the library insert was obtained by dideoxy-nucleotide sequencing with primers that hybridize to either side of the BamHI site in the tetracycline- resistance gene of YCpSO (see pBR322 primers #1219 S1-d(ATGCGTCCGGCGTAGA)-
3' and #1223 5'-d(CACTATCGACTACGCGATCA)-3' from New England Biolabs).
Sequence information was found in the Yeast Genome Database using the Basic Local Alignment Search Tool (BLAST) (Altsçhul et al., 1990). Yeast whole-cell extracts The preparation of yeast, whole-ceU extracts is described in Chapter 2. These extracts were used for in vitro, non-specific transcription assays (described in Chapter 2), for in
vitro, promoter-specific assays (described in Chapter 2) and for in vitro splicing assays (see below).
Zn vitro, yeast whole-cell-extract splicing assays
Capped and 32~-labelledpre-mRNA was synthesized from plasmid pYF1122 DNA (Lin et al., 1985) using T7 RNA polymerase as follows. pYF1122 plasmid DNA (2 pg)
digested with EcoRI was transcribed by T7 RNA polymerase for 1.5 hrs at 23°C in 18.5 pl containing 30 U T7 RNA polymerase (Pharmacia), 40 mM Tris-HCI (pH 7.3, 6 mM MgC12, 10 mM NaCI, 2 m.spennidine, 10 mM DTT, 1 mM each of ATP, GTP, CTP, 50 ph4 UTP (Pharmacia), 0.9 mM ~~G(S')~~~(S)G(Pharmacia), 50 pCi [a-32~1-UTP (3000 Ci/mmol; Mandel), 16 U RNasin (Promega). The reaction products were purified using RNeasy spin columns (Qiagen) according to the directions of the manufacturer and were eluted in a total of 50 pl of distilled, RNase-free water and stored at -20°C. In vitro splicing reactions (modified from (Lin et al., 1985; Newman et ai., 1985)) were carried out in a total volume of 10 pl containing 60 mM potassium phosphate (pH 7.3, 3 mrM MgC12, 3% poly-ethylene glyco1(4000), f mM ATP, 0.4 U/pl RNasin (Prornega), 0.2 pl of capped, 32~-labelledpre-actin mRNA from the above reaction (2-6 kcpm according to a BIOSCAN Tabletop counter) and 4 pl of whole-ce11 extract (20
pg/p1). The reaction was usuaily started by adding extract and was incubated at 23°C for 7 to 21 min. The reaction was stopped by addition of 2 pl of stop solution (1 mghl proteinase K, 50 mM EDTA and 1% SDS)and incubating at 37°C for 15 min. 200 pl of a cocktail containing 50 mM sodium acetate (pH 4.8), 1 mM EDTA. 0.1% SDS and 25 pg/d E. coli tRNA) was added and protein was extracted with phenol:chloroform:isoamyl alcohol (2524: 1). RNA was ethanol-precipitated, washed with 70% ethanol, dried in a speed-vacuum and finally dissolved in 17 pl IXTE and 23 pl of loading buffer (90% formamide, 1X TBE buffer, 2.5 mgmi bromophenol blue and 2.5 mg/d xylene cyanol). Samples were heated to 65°C for 5 min. before loading 20 pl on a pre-run, 0.8 mm, 6% poiyacrylarnide/0.2% bisacrylamiden M urea gel. Electrophoresis was at 350 v for 2 hours. The gel was dried at 80°C for 30 min. and then exposed to a Molecular Dynamics phosphor screen for 6 to 12 hrs.
Purification of His-tagged Gcr3p €rom yeast A gcr3::HIS3 disruption strain (YF2330) was transformed with plasmid pYF1771 (YCpSO with HispHA-GCR3) to generate YF2334 or with pYF1693 (YCp5O with
GCR3) to generate YF2333. A 1L culture of each strain was grown to an OD6w of 2 in
SC-(uracil, histidine) and harvested by centrifugation. Whole-ceil extracts were prepared as described above except that buffer EB lacked DTï and following concentration with ammonium sulfate, extracts were resuspended in and dialyzed against buffer NMDB (20 mM HEPES pH 8.0, 50 mM imidazole, 20% glycerol, 10 mM MgS04, 10 mM PME, 1 rnM PMSF, 5 pg/rnl pepstatin, 10 mM benzamidine, 5 pghl leupeptin) with 1M NaCI. Protein (90 mg) was combined with 1 ml of nickel-beads (Qiagen) (pre-equilibrated in NMDB with 1M NaCl) and rnixed end-over-end at 4°C for 1 hour. Beads were loaded ont0 a 10 ml Quick-Prep column and washed twice with 4 voLumes of 1M NaCl in NMDB and then with 4 volumes of mi3 alone. Protein was eluted with 4 elutions each with 0.5 ml of 0.25 M imidazole in NMDB. Eluate was dialyzed against TDB (see above) over night. Conductivity of the eluate following transcription was equal to TDB buffer alone. Samples were stored ai -70°C. A single 100 kDa band was detected by protein-blot analysis with anti-HA antibody (12CA5) in the first two elutions. CTD pull-down experiments Whole-ceil yeast extract (described in Chapter 2) was prepared from yeast YHU3003- 2B transformed with plasmid pYF1720 (HA-GCR3, LEU2, 2-pî4). GST and GST- fusion proteins were prepared from E. coli using glutathione-sepharose chromatography (Phmacia Biotech) according to the manufacturer's instructions. Plasmids encoding GST-CTD and GST-AS were a gift from Jeff Corden and are described in (Patturajan et al., 1998). GST-CTD and GST-AS were phosphorylated using HeLa extract as described in (McCracken et al., 1997); 200 pg of fusion protein was incubated at 30°C for 40 min. in 200 pl containing 20 mM HEPES (pH 7.9), 10 mM MgC12, 2 mM Dm, 2 rnM ATP, 20 rnM creatine phosphate, 50 pghl creatine kinase, 2 pg/d bovine serum albumin, 20 mM p-glycerophosphate, 2 pM microcystin (Calbiochem) 2 mM benzamidine, 0.01 pg/pl aprotinin, 0.01 palpepstatin, 0.02 pglpl leupeptin, 80 ph4 PMSF and 0.8 mg/d HeLa extract (Promega). Reactions were placed on ice before coupling to glutathione beads. Glutathione beads were pre-blmked in ACB buffer with 4 mg of YF2070 yeast extract per ml of beads. GST or GST-fusion proteins (200 pg) were bound to 40 pl of glutathione-sepharose beads in ACB 100 buEer(20 mM HEPES (pH 7.9), 0.1 mM EDTA. 1 mM DTT, 20% glycerol, 0.5 LM rnicrocystin, 1 mM glycerophosphate, 0.1% NP40 with 100 rnM NaCl) for 30 min. on ice. Beads were washed with 10 volumes of ACB with 1M NaCl followed by 10 volumes of ACB 100. Beads were then combined with 200 pg of yeast extract (see above) in ACB 100 at 4°C for 1 hour with end-over-end mixing. Beads were washed 4 times with 500 pl of ACB 100 over a 15 min. time period Elution was with 160 pl of ACB with 1M NaCI. For guanylyl-transferase assays (Shuman, 1982; Shuman et al., 1994), 50 pl of each elution was dialyzed ovemight in GT buffer (20 mM HEPES (pH 7.9), 50 mM KCl, 0.2 rnM EDTA, 0.5 mM DTT and 20% glycerol). Assays were in a total volume of 20 pl containing 15 pl of column eluate, 50 mM Tris-Cl (pH 8.0), 10 mM MnC12 and 10 pCi [,-32~]-GTP (3000 Ci/rnM; Mandel). Reactions were at 37OC for 5 min. and were stopped by adding 5 pl of SDS-PAGEloading buffer. Formation of the labelled 5 1 kDa GMP-Ceg 1p was detected by autoradiography after SDS-PAGE.
Antibody pull-down assays Whole-ce11 extracts were made from yeast YF2070 or YF2070 transformed with pYF1707 (HA-Gcr3p). Extracts (800 pg) were combined with 9 pg of antibody (8WG 16 or 12CA5) in a total volume of 500 pl containing IP buffer (50 mM potassium acetate, 10 mM HEPES (pH 7.6), 1 mM DTT, 1 mM EDTA, 10% glyceroi, 0.2 mM PMSF, 5 pg/d leupeptin, 1O m.benzamidine, 10 pg/ml aprotinin, 3.5 pghl pepstatin).and mixed for 1 hour at 4°C. Protein-A sepharose beads (Sigma; 20 pl of a 1: 1 slurry in IP buffer) were added and rnixing was continued for 1 hour at 4°C. The beads were washed three times with IP buffer containing 50-200 mM potassium acetate and were finally resuspended in 50 pl of 1X SDS-PAGE buffer. One third of this sample was electrophoresed on a SDS, polyacrylamide(7%) gel and blotted to PVDF. The membrane was blocked and probed as described in Chapter 2 using either a 1: 10,000 dilution of 12CA5 (anti-HA) or a 1:5000 dilution of 8WG 16 (anti-CTD). Secondary antibody was a 1: 15,000 dilution of goat, anti mouse IgG antibody coupled to horse radish peroxidase in both cases. The blot was developed using a LumiGlo Chemiluminescent substrate kit (Kirkegaard and Perry Laboratories) according to the manufacturer's instructions. The CO-irnmunoprecipitation experiment was repeated using whole-ce11 extracts made from yeast YHU3003 transfoxmed with pYF1721 (same as pYF1720) with sirnilar results. Wcsults
Thc yt;rst strain used to screen for synthetic-lethd mutations is shown in Figure 3.2.
ï?li\ syçtem is bmed on the ability of ade2, ADE3 yeast cels to accumulate a red pigment uhik trde2. ade3 ceiis do not (Bender and Pringle, 1991; Jones and Fink, 1981). The starting strain carries a mutation (rpo2I -30) on the chromosome. This mutation by itself is not lcthal to the ceIl at 30°C but prevents growth at 37°C. A maintenance plasrnid carries a
WT copy of RPOZl which is not required for growth at 30°C and cells grown at this temperature cmspontaneously lose this plasrnid. As a result, colonies which arise frorn this strain grown on nch medium at 30°C contain a mixture of celis; those that have this maintenance phsrnid have an ade2, ADE3 phenotype (red) and those that have lost the plasrnid have an ade2. ade3 (white) phenotype. This gives the colony a red and white sectored appearance or what 1 will cda se& phenotype. Secondary mutations were created in the starting strain using UV mutagenesis. Cells were mutagenized to a level at which only 10%of the starting cells survived. These cells were spread to rich agar medium (YPD), incubated for 4 to 5 days at 30°C and were screened for colonies that were completely red (Le., sect-). These colonies are candidates for carriers of secondary mutations that are lethal in combination with the rpo2I-30 mutation. Cells that spontaneously lose the RPOZI, ADE3 maintenance-plasmid die and the resulting colony is composed entirely of ade2, ADE3 (red) ceUs and appears to be sect- Approximately 30,000 colonies were screened in this manner and a total of 9 1 putative synthetic-lethals (sect-) were colkcted. Many of these were either just slowly sectonng or false sect- colonies since they sectored after king re-streaked to YPD a second or third time. These were elirninated, leaving 44 candidates. Several cIasses of mutations may cause a sect- phenotype but are not relevant to this study. For example, conversion of the chromosomal ade3::HisG locus Figure 3.2: Synthetic-lethal screen method. A. Schematic of strain YF2079 used to isolate mutations that are synthetically lethal in combination witb rpoîi-30. Introduction of secondary mutations that are syntheticaily lethal will not aliow the ce11 to lose the RP021 maintenance plasmid (pYF1465) and remain dive. Colonies arising from these ceils then must maintain the plasmid and are therefore red (ade2 ADE3) and appear to be non- sectoring and are sensitive to 5-FOA (since they obligatorily carry the UM3marker). B. Sectonng can be restored by supplying a WT copy of RP021 on pYF1S 13. rpo21a -
sporrtaneaus loss d plasmid leads to âeath of cell at WC cdoriies appear sectg and are FOAS
B. Introôudon of mather WT RP021 pksmid alkws th. RP021-AOE3 ph^# t~ b. bd
X-
rpo2 1 -30-
UR93 ADE3
spcmtaneous bss of piamid is to(eraMe cdoriies appear sed+ and are FOAr to ADE3 would cause cells to become red constitutively regardless of the presence or absence of the ADE3, RPO2l maintenance plasmid. For this reason, synthetic-lethal mutants were assayed for their inability to lose the plasmid by an independent assay. The maintenance plasmid also carries a URA3 marker; therefore, cells unable to lose the maintenance plasmid would be 5-FOA sensitive (Boeke et al., 1987). Putative mutations that were 5-FOA resistant were discarded. This left eight probable mutants. The large number of mutants eliminated at this stage probabiy included strains that were able to lose the ADE3, RP021 plasmid but gew much slower in the absence of the plasmid and therefore produced colonies that appeared to be sect- on YPD. Other mutations that might cause a sect- phenotype include those that confer a requirement for some element of the maintenance plasmid other than RP021 or that reduce the ability of the cell spontaneously to lose the plasrnid. Altematively, the plasmid itself might recombine with the chromosome, making loss of the ADE3 and URA3 markers impossible. Mutants belonging to either of these classes were eliminated by transforming with a plasrnid unrelated to the maintenance plasmid except for the presence of a RPO2 I gene (pYF15 13). Mutants that were unable to lose the maintenance plasmid under these conditions were discarded leaving five potential synthetic-lethal mutants (see Table 3.3). Finally, sect- colonies might arise from secondary mutations in the chromosomal copy of t-po21-30. These potential intragenic mutations were identified by re-introducing the original rpo21-30 allele on a plasmid. Three of the remaining five mutants were able to sector when transformed with thjs plasmid. These sect- strains then are Iikely to carry additional mutations in the chfomosomai pu21-30 gene. Altemativety, these sect- strains may carry second-site mutations that are synthetically lethal with rpo21-30 but for some reason this lethality can be complemented by adding back extra copies of the rpo21-30 allele. In any case, these were not studied Mer. Table 3.3: Synthetic-Iethal screen results strain mutant sectt/' 5-FOAS sect+/' se&/' sectt/- 37°C sect+/- 2:2 number RP021 rpo21-30 RP022 diploid
YF2099 Pl1 s + + + rd rd YF2102 P21 s + t + nd nd YF2105 P47 s + - + Y es YF2108 P56 s + + + nd nd YF2111 P88 s + - slow + Yes notes: a Five potential mutants from the synthetic-lethal screen are shown. Ability of mutant to wtor on YPD agar medium incubated at 30OC after 4 days. Ability of the mutant to grow on 5-FOA medium incubated at 30°C. Ability of the mutant to sector on YPD medium after introduction of plasrnid pYF1513. Ability of mutant to sector on YPD medium after introduction of plasmid pYF1550. Ability of the mutant to sector on YPD medium after transformation with an RP022 plasmid. g Ability of mutant to grow on YPD agar medium incubated at 37OC. Ability of the corresponding diploid to sector (see text). Ability of the sect- phenotype to segregate 2:2 (see text). Two sect- strains remained that could not be complemented by rpo21-30. These mutants (P47 and P88) were assayed for phenotypes. Strains YF2105 and YF2111 were spread on YPD at various temperatures and observed for growth defects with respect to the WT strain YF2079. P47 was slow-growing at 30°C and dead at 37°C. P88 was slow- growing at both 30°C and 37°C. Since these mutants carried WT copies of RP021,the observed growth defects must be caused by some second-site mutation, potentially
associated with the sarne mutation that was synthetically lethal with rpo2l-30. Both of the synthetic-lethals were shown to be recessive. P47 and P88 were crossed to YF2097 (MA Ta rpo21-30 ade2-1 ade3::HisG). The resulting diploids were sect+ and FOAr indicating that the P47 and P88 mutants were recessive. The diploids were sporulated and the dissected tetrad products were assayed for their sectoring phenotype and for the growth defects at 30°C and 37°C. The sect-:sect+ phenotype segregated 2:2 in the complete tetrads that were obtained indicating that the synthetic-lethality observed in mutants P47 and P88 was due to a single locus in combination with rpo21-30. Furthemore, sect- spore products from both crosses were found to CO-segregatewith the parental temperature sensitivity/slow growth phenotypes of P47 and P88 respectively. This indicated that the loci responsible for the synthetic-lethality of P47 and P88 were likely to be responsible for the associated growth defects. These associated defects are useful in screening for the WT copies of P47 and P88 from a yeast genomic library (see below).
Rescue of P47 and PSS synthetic-lethality The ability of various WT and mutant alleles of PO21 to rescue the synthetic-lethality of P47 and P88 was assayed. Yeast strains YF2133 (P47) and YF2134 (P88) were transformed with the plasmids listed in Table 3.4 and were assayed for their ability to sector on YPD medium at 30°C. The allele of RP021 carried by eacb of these plasrnids, the associated phenotype and the region of RPO2l in which the mutation resides are indicated in Table 3.4. Plasmid pYF15 13 carrying a WT allele of RP021 was able to rescue the sect- phenotype of both P47 and P88. However, plasmids that were identical to pYFl5 13 but carried mutations in the RPO21-ZBD were unable to restore sectoring (for example, piasmids pYF1547 to 1550). Furthemore, while RP021 on pYF 1409 was able partially to restore sectoring, versions of the plasmid carrying mutant rpo21 alleles were unable to restore sectoring. This suggests that the synthetic-lethality observed in P47 and
P88 is neither specific to rpo21-30 nor to mutations in the ZBD. In theory, either P47 or P88 could have been identified in synthetic-lethal screens starting with any one of the rpo21 mutant alleles listed in Table: 3.4. For this reason, 1 expected that whatever was learned about P47 and P88 would not necessarily be related specifically to the functional role of the Rpo2 lp-ZBD itself. However, these results did not rule out the possibility that the screen had reveded other components of the transcriptional machinery. Rescue of the P47 and P88 sect- phenotypes were also assayed at 23°C (data not shown). Generally the results were the sarne as for 30°C with a few exceptions. Plasmid pYF1549 (rpo21-29)was reproducibly able to rescue P47 at 23°C although it couId not restore sectoring to P88. Furthemore, pYF1548 (rpo21-28) was able to rescue P88 at 23°C aithough it was unable to restore sectonng to P47. The significance of this result is uncertain but may be of use later on (see Discussion). Also, pYF1410 (rpo21-5) was able partially to restore sectoring to P47 at 23°C. It was surprising that the thme WTRP021 plasmids used above were different in their
abilities to rescue the synthetic-lethal phenotypes (Table 3.4, plasmids pYF 15 13, pYF 1562 and pYF1409. Each of these plasmids was re-examined for its ability to support growth in an rpoZi:.-HIS3 gene disruption strain (see YF2380 (pYF1409), YF2382 (pYF 15 13) and YF2384 (pYF1562). Growth was compared among the three strains on YPD medium. As expected, no differences were observed at 30°C- However, pYF1409 was unable to support WT-like growth at 23°C or lower. This phenotype has not been noted previously Table 3.4: Summary of synthetic-lethal rescue experiments scct+/- sect+/- plaçmid allele phenoty pe regione P47 P88 (Y F2133) (YF2134) i ~YF1513~ RP021 WT + t p~~1562b RP021 WT t - ~YF1409~ RF021 cs at 23°C +/- +/- pYF1547" rp02 1-2 7 (C67S) sg at 37°C ZBD - - ~YF1548~ rpo21-28 (C70S) sg a t 37°C ZBD - pYF1549" rpo21-29 (C77S) t s ZBD - pYF1550a rpo2 1-30 (H80Y) t s ZBD -
~YF1414~ rpo2 1-8 t s N-term - pYF141W rpo2 1-5 t s A-B - p~~15ti3b sit 1-4 ts F na pYF1411C rp02 1-4 t s F-G - pYF1413C rpo2 1- 7 t s G-H - - p~ ~1545~ rpo21-RI 1 CS CTD - -
Notes and refewnces to Table 3.4 a pYF1513 is parental to pYF1547-1550 (described in Chapter 2) pYF1562 is parental to pYF1563 (described in (1)) pYF1409 is parental to pYF1410 to pYF1414 (described in (2)) pYF1545 (aka JA501) carries a mutant allele of RP021 that has only eleven CTD repeats. This plasmid is based on pJAYlOl (aka pYF1544) and was constructed by Xia0 Hua. ZBD: zinc-binding domain; A-H: see (3); CTD: C-terminal domain
References 1, Archambault, J., Jansma, D. B., Kawasoe, J. H., Arndt, K. T., Greenblatt, J., and Friesen, J. D. (1998) 1 Bacterio! 180(10), 2590-8 2. Archarnbault, J., Drebot, M. A., Stone, J. C., and Friesen, J. D. (1992) Mol Gert Getiet 232(3), 408-14 3. Jokerst, R. S., Weeks, J, R., Zehring, W. A., and Creenleaf, A, L. (1989) Molecitlsr G. Getiernl Ge~ielics215(2), 266-75 and may explain why this plasrnid was unable Mly to rescue the sect- phenotype of P47 and P88. It is unclear why pYF1562 is unable to rescue P88. The three plasmids differ at least in the chromosomal fragments of RPOZI that are subcloned. The plasmid backbone aiso differs between pYF1409 and the other two (see Figure: 3.1). in addition, the source of the RP021 may be different in each case, although 1 was unable to trace the RPOSI source for each plasmid. In any case, one possible explanation is that different WT RPO2i plasmids express different levels of steady-state Rpo21p and these levels differ in their
ability to cornplement the synthetic-lethality between rpo21-30 and P47 or Pû8.
Steady-state levels of Rpo2lp in P47 and P88.
The effect of P47 and P88 on Rpo2lp steady-state leveis was examined. Protein-blot
analysis using 8WG16 antibody was used to detect steady-state levels of RpoZlp in strains that carried either P47 or P88 and a WT, chromosomal RP021 (see Figure 3.3). No significant decrease was seen in Rpo2lp levels compared to WT. This indicates that the observed synthetic-lethality is probably not due to a decrease in expression of the rpo21-30 (H8OY) dele.
Identification of P47 as an allele of SRBS Strain YF2 133 (P47) was transformed with the CEN BANK A library (see ATCC #37415). This library carries a partial Sau3Al digest of yeast genomic DNA subcloned into the BarnHI site of the plasrnid vector YCpSO (URA3, CEN4, ARSl) (Thrash et ai., 1985). Plasrnids carrying the genes responsible for the synthetic-lethality of P47 were screened for by selecting transformants that were able to grow like WT at 37°C and that restored sectoring at 30°C. Ten transformants of YF2133 were found that grew like WT at 37°C on SC-(uracil, Ieucine) medium. When streaked to YPD medium at 30°C seven of these were able to Figure 3.3: Rpo2lp steady-state levels in srb5-IO0 and gcr3-100 mutant
strains. Yeast whole-ce11 extracts were examined for the presence of Rpo2lp using
antibody (8WG 16) that recognizes the C-terminal domain of Rpo2 lp. Six pg (lanes 1,4,
and 7), twelve pg (Ianes 2,s and 8) and eighteen pg (lanes 3,6, and 9) of each extract was
assayed. Rpo2 lp signal was quantified for each. No significant ciifferences were found between the WT (YF2094)strain and those carry ing mutant alleles srb5-100 (YF2344) or gcr3- 1O0 (YM349).
sector. These seven transformants were also 5-FOA resistant at 30°C but were sensitive at
37°C. These data indicated that suppression of the ts and sect- phenotypes in these seven transformants was plasmid-dependent and that the plasrnids were carrying the WT version of the P47 gene. The remaining three transformants were either chromosomal or higher-
copy suppressors of the P47 ts phenotype. Loss of the maintenance plasrnid (pYF 1466) from these transformants was screened for by selecting white colonies on YPD. The remaining library plasmids were rescued by preparing DNA from each transformant and transfonning into E culi. Restriction-enzyme
analysis of these library plasmid DNAs with EcoRV indicated that each subclone was identical. Sequencing analysis of the insert DNA in one of these (pYF1494) using primers ID03 and ID02 indicated that the insert encompassed 13.6 kb extending from bp 699797 to 702365 on chromosome VII. This fragment contained at least 10 open reading frames (ORFs) including SRBS, a known component of the RNA polyrnerase II holoenzyme (Thompson et al., 1993). SRBS sequence was amplified from the library insert and subcloned to the plasmid pRS315 to generate three independent subclones; pYF1573- 1575. Each of these subclones was able to complement the sect- and ts phenotype of P47(YF2105). This complementation, taken together with the fact that SRBS from YF2 105 had a frarne-shift mutation (see Chapter 4), allowed me to conclude that P47 was allelic with SRBS and that a mutant allele of this gene was synthetically lethal with rpo2l- 30. The identification of a known RNAP II holoenzyme component validated the ability of the synthetic-lethal screen to identify new components of the transcriptional machinery.
Identification of P88 as an allele of GCR3 Strain YF2134 (P88) was also transforrned with the CEN BANK A library (see above). Plasmids that potentially carried the WT version of P88 were screened for by selecting for faster (WT-like) growth on SC-(uracil, leucine) medium at 37°C. Fast- growing transformants were plated on YPD at 30°C and screened for the ability to sector. Two such transformants were found. The Library plasmid DNA was prepared from these and transfonned into E. coli. Re-transformation of each plasmid (pYFl6 18 and pYF 1620) into YF2 134 confmed that rescue of the slow-growth and sect- phenotypes were plasmid- dependent. Partial sequence andysis of pYFl6 18 mapped the ends of the 1 1,111 bp library insert to nt 5 10,194-52 1,304 of chromosome XIII. This fragment encompassed two open reading frames; YMRl24W (unknown function) and STOUGCR3 (Largest subunit of the pre-mRNA, cap-binding cornplex). A BamHI site in the GCR3 ORF
(unique to the pM1618 library plasmid) was used to create a frame-shifi mutation by end- filling. The resulting plasmid (pYF1672) was unable to restore WT-like growth or sectoring to YF2134. Furthermore, restriction analysis revealed that both complementing library plasmids shared a 4.2 Kb ClaI fragment that encompassed only the GCR3 ORE This fragment subcloned to YCpSO (pYF1693) was able to restore both WT-like growth and sectoring to YF2 134. 1 concluded that P88 is allelic with STOUGCR3. The new P88 aüele of GCR3 was designated gcr3-la).
It is likely that GCR3 is P88 itself, and not a high-copy suppressor of the P88 mutation in some other gene besides GCR3, for the following reasons. GCR3 was able to complement both the temperature-sensitive and the sect- phenotype of P88. Furthemore, GCR3 on a high-copy plasmid (pYF1720) was unable to suppress the temperature- sensitive growth defect of rpo2 1-30 when introduced into yeast strain YF2 145 (see Table 2.2). Finally, a single-nucleotide substitution mutation was found in the GCR3 ORF of the P88 mutant strain (see next section).
Mapping and identification of the mutation in GCR3 The GCR3 ORF is 2968 bp. The position of the mutation in gcr3-100 was first narrowed down to a smailer region by gap-recombination. The plasmid pYF172O (HA- tagged GCR3 subcloned to YEp3S 1; LEU2, 2-CLM)) was digested with different sets of restriction enzymes that cut only inside the ORF of GCR3. This set of plasmid digestions was introduced into yeast strain YF2 1 11 (aka P88; rpo21-30, pYF1465 (RPOZI, ADE3, 2-phd,LIRA3). Transformants in which the gap had been repaired using the chromosomal sequence as template were selected for on SC medium lacking uracil and leucine. These transformants were then assayed for sectoring on YPD medium. Plasrnid gaps that were repaired by WT, chromosomal sequence would allow for the loss of the RPO2 1
maintenance plasmid (set+). lf the gap was repaired with a region of chromosomal DNA
that contained the P88 mutation, then the transformant would be sect-. Figure 3.4 shows that pYF1720 digested with NdeI and BgZII (removing most of the ORF) was unable to restore sectoring, indicating that the chromosomal mutation was positioned somewhere between these two sites. However, gaps created by either NdeI or Bgm aione were able to restore sectoring indicating that the mutation did not lie within these gaps. This left the possibility that the mutation was between nucleotides 1128 and 2327 (see Figure 3.4). DNA between these sites (1.2 kb) was amplified directiy from the chromosome and sequenced on both strands- Sequence analysis reveafed a single C to T transition at nucleotide 1970 (where the A of the initiating ATG is +1) thereby changing a glutamine codon to a tennination codon after amino acid 523 of a 861 amino acid protein (Figure 3.4). This change was confimed by restriction-enzyme analysis since the mutation creates a new Dra1 site. The identification of the mutation in the GCR3 ORF together with the fact that the synthetic-lethaLity/temperature sensitivity of the original strain
CO-segregated2:2 confmed that the true synthetic-lethal gene had been cloned. Figure 3.4: Identfication of the gcr3- 100 mutation. Plasmid pYF 1720 digested with various restriction enzymes was introduced into yeast strain YF2 1 1 1 to assay for its ability to restore sectoring after king repaired using chromosomal sequence as template. Sectoring was not restored if sequence between nucleotides 1128 and 2327 of the GCR3
ORF was repaired. DNA between these coordinates was amplified from the chromosome of YF2 11 1 and the sequence was anaiysed. A single C to T transition was detected when compared to DNA ampiified from a WT source (YF2070). This changes codon 524 to a stop codon and the resulting protein is tmncated to 523 amino acids from 861 in the WT. Ndel / @Ill
NcoV Bglll
SpeV Ndel
Ndel
Bglll
expect ed location
TTT GCT AAA AAT
TTT GCT AAA AAT TTG ATT AAA GAA CTA Oral
Ocr3-1 OOp FAKNL gcr3-100 confers a defect in in vitro, promoter-specific transcription The gc7-3-100 strain (YF2111) was back-crossed to a strain carrying WT alleles of both RPOSI and GCR3 (YF2094). Spore products carrying only the gcr3-100 mutant allele were selected (see YF2349 and YF2350). The slow-growth phenotype of these spore products was complemented by a plasmid canying a WT copy of GCR3 but not by a control plasmid without an insert or carrying RP021. Two independent mutant extracts as well as two WT controls (YF2094) were prepared for the experiments listed below. Equal amounts of gcr3-100 and WT extracts (as determined by Bradford assays) were assayed for promoter-dependent transcription activity. Activity present in gcr3-100 extracts was only 60% of that in WT extracts (Figure 3.5). These assays were performed using amounts of extract and reaction times that were non-saturating (data not shown). These extracts were also tested for prornoter-independent transcription activity and were found to have activity similar to WT levels (Figure 3.6). This is the same pattern observed for other transcription factors such as SRBS, which are required for eficient promoter- specific transcription initiation and template cornmitment but have no effect on promoter- independent transcription. Before proceeding further, 1 wanted to eliminate the possibility that a mutation in gcr3- 100 might alter the stability of nascent transcripts, thereby leading to a decrease in activity. Capped, actin pre-mRNA was made in vitro with T7 RNA polymerase and added to either WT or mutant extract. Sarnples were taken at 5 min. and 1 hour and exarnined by denaturing polyacrylamide gel eIectrophoresis. Quantification revealed no significant difference in the levels of mRNA recovered from the two extracts (data not shown). In a separate expriment, promoter-directed transcription reactions were performed using either WT or gcr3-100 extract. Reactions were stopped after one hour by adding a-amanitin.
Sarnples of these reactions were then taken immediately or after an additional 1 hour incubation at 23°C (during which time transcripts that were aiready made could be degraded). Promoter-specific transcription products were examined by denaturing gel Figure 3.5: Extracts of gcr3-100 have an in vitro promoter-directed transcription defect. Panel A: Template DNA pGAL4CG- is a G-less cassette used to assay prornoter-specific transcription activity in whole-ce11 extracts. The CYC 1 promoter drives basal-level transcription that may be enhanced by adding the transcriptionai-activator pmtein GaWp. Transcripts initiated from this promoter do not incorporate guanosine and so are not depded by the presence of RNase T 1. Labelled transcripts of 375 and 350 nt are detected
by autoradiography following denaturing polyacrylamide gel electrophoresis. Promoter-specific activity of 4O,8O and 180 pg (determined by Bradford assays) of either WT (YF2094) or gcr3-100 (YF2349) extracts are shown. These experiments were
repeated using two independent sets of extracts. Panel B: Quantification of in vitro promoter-specific transcription activity in WT and p-3-la) extracts. Five reactions were carried out for each extract at two temperatures. In each case, 80 pg of extract was used and reactions were for I hour. Prornoter-specific transcription products were detected using a phosphor screen (Molecular Dynamics).
Quantification of the data was with image-Quant software (Molecular Dynamics). Data are normalized to activity in the WT GCR3 extract at 23°C.
Figure 3.6: Extracts of gcr3-100 have non-specific RNA polymerase II
activity equivalent to that of WT extracts. Whole-ce11 extract (20 pghssay) from WT (YF2094) or gcr3-100 (YF2349) was assayed for non-specific transcription activity using calf-thymus DNA as template. Al1 reactions were at 23°C for 20 min. and were repeated for two independent sets of extracts. Combined activities from RNA polymerase 1 and IIi were measured in the presence of a-arnanitin and were subtracted from total RNA polymerase activity (no a-amanitin present) to obtain RNA polymerase II activity. The bars for each mesurement represent the average of three trials where the standard deviation was less than 5%. For each extract, the RNA polymerase II activity is represented by a black bar (measured as a percentage of total activity in the extract) and by a stippled bar
(measured as a percentage of RNA polymerase II activity present in an equivalent amount of WT extract). The combined activities of RNA polymerases I and III are shown by the gey bar expresseci as a percentage of RNA polymerase 1 and IIi activity in WT extract. pol II with respect to total (%) pol II with respect to M/T (%) a pol I+III with respect to WT (%) electrophoresis and quantified using Image-Quant software. No difference in the rate of
transcnpt degradation was observed between the WT and gcr3-100 extract transcripts over the one hour incubation period at 23°C (data not shown). I concluded from these experiments that the differences observed in promoter-specific transcription activity between the WT and gcr3-100 extracts (see Figure 3.5) was not due to enhanced
degradation of transcription products in gcr3-1Ml extracts.
Next, a GCR3-deletion strain was examined for a similar transcription defect. GCR3 is a non-essentiai gene and extract made from a deletion strain approximates the effect of depleting Gcr3p from a WT extract. Such an extract can be used for a Gcr3p addback expriment to determine whether the protein has a direct effect on transcription.
The GCR3 ORF was replaced by the HIS3 ORF as a marker. The gcr3-deletion strain had a slow-growth phenotype at both 30°C and 37°C similar to that conferred by gcr3-100. Extracts were made from two independent WT and two deletion strains. No significant
difference was found between WT and Aga3 extracts for promoter-dependent transcription
(Figure 3.7 ). It is possible that the transcription defect observed for the gcr3-100 extract may be due specificaily to this allele. Gcr3-100 protein may form an aberrant or non- functional complex that interferes with transcription. Cap-binding complex (CBC) irnmuno-purified from yeast was obtained from hri Fortes and Iain Mattaj (Lewis et al., 1996b) in order to determine if it would restore transcription activity in a gcr3-100 extract. 1 chose to add both subunits of the CBC (Gcr3p and Mudl 3p) to gcr3- 100 extracts because a GCR3-deletion strain lacks Mudl 3p
(personal communication in (Colot et al., 1996)). This preparation was able to restore partially the formation of the splicing cornmitment complex in CBC-irnrnunodepleted extracts (using 0.2 pg CBC per 40 pg of extract; Puri Fortes; personal communication). Also, this preparation had cap-binding activity in a gel-mobility shift assay (Puri Fortes; personal communication and data not shown). This CBC preparation had no restorative effect on promoter-directed transcription activity in gcr3-100 extracts (using 0.2 to 0.8 pg of CBC per 80 pg of yeast extract; data not shown). These results argue that the promoter- directed transcription defect of gcr3-100 extracts is an indirect one. Altematively, WT CBC may be unable to compte with mutant gcr3-IO0 protein present in an endogenous non-functionai complex. Thus, it is not clear if Gcr3p plays a role directiy in transcription. To investigate the possibility that the effect of Gcr3p on transcription is direct or indirect, 1 performed experiments to determine if it is associated with RNA polymerase. Given the recent studies
which have shown that the process of transcription and mRNA splicing might be coupled, it seemed plausible that Gcr3p might be a component of the holoenzyrne. However, two separate experiments revealed that this is probably not the case. First, a monoclonal antibody that recognizes the C-terminal domain of Rpo2lp was unable to co- immunoprecipitate an HA-tagged Gcr3p from yeast extract, nor was anti-HA able to co- immunoprecipitate Rpo2 lp (Figure 3.8). Second, it is possible that Gcr3p interacts with the phosphorylated CTD of Rpo2lp. Such an interaction would not have been detected in the above assay since the anti-CTD antibody used recognizes the non-phosphorylated form (Patturajan et al., 1998). To address this possibility, phosphorylated GST-CTD fusion protein was bound to glutathione beads. Ttiese beads did not pull down HA-Gcr3p from a yeast extract, dthough they were able to pull down one component of the capping complex (guanylyl- transferase) under the same conditions (Figure 3.9). Figure 3.7: Extracts of Agcr3 have WT levels of promoter-directed transcription. Quantification of in vitro promoter-specific transcription activity in WT (YF2094) and gcr3::HIS3 (YF2330) extracts. Five reactions were camed out for each extract. In each case, 80 pg of extract was used and reactions were for 1 hour at 23°C
Promoter-specific transcription products were detected usïng a phosphor screen (Molecular Dynamics). Quantification of the data was with Image-Quant software (Molecular Dynamics). Data are normalized to activity in WT extract. This assay was repeated for two independent sets of extracts for each strain with similar results.
Figure 3.8: HA-tagged Gcr3p does not CO-immunoprecipitate with RNA polymerase II Panel A shows the rationale of the pull-down expriment. Antibodies against either the CTD of Rpo21p (8WGL6) or against the HA epitope fused to Gcr3p (12CA5) were added to yeast whole-ceIl extract. Antibody and any associated proteins were isolated from the extract using protein-A sepharose beads. Proteins associated with the antibodies were anaiyzed by blotthg to hobilon membrane and probing with either anti-HA or anti-CTD antibody. Panel B: Protein A beads alone or in combination with anti-HA or anti-CTD antibodies were incubated with extract that carried HA-tagged Gcr3p. Beads were washed with buffer containing 50, 100 or 200 rnM potassium acetate then precipitated proteins were analysed by western blotting. Anti-Cm antibody (a--) was able to irnrnunoprecipitate Rpo2lp (lanes 3, 6, 8) but not HA-Gcr3p. Anti-HA antibody (a-HA) was able to pull down HA-tagged Gcr3p and Rpo2lp under low salt conditions (lanes 2 and 5) however, this association was non-specific since anti-HA was also able to pull down Rpo2 lp from extract that did not contain HA-tagged Gcr3p (Panel C; lane 5). Panel C: Protein A beads alone (beads; Ianes 1 and 4) or in combination with antibody that recognizes the HA-epitope of HA-tagged Gcr3p (a-HA;lanes 2 and 5) or the CTD of Rpo2lp (a-CTD;lanes 3 and 6) were incubated with yeast whole-ce11 extract containing either HA-tagged Gcr3p or untagged Gcr3p. Each of the two antibodies was able to pull down its cognate protein as indicated by protein-blot analysis (Ianes 2,3 and 6). Anti-CTD was unable to pull down HA-tagged Gcr3p (lanes 3 and 6); however, anti-HA antibody was able to pull down Rpo2lp (lane 2) but this interaction did not involve HA-tagged Gcr3p, since it did not rely on the presence of the HA-epitope in the extract (lane 5). CTD anti-CTD
HA-GcBp (100 KDa)
HA-Gcr3p (1 00 KDa)
Rpo2l p . . ant i-CTD (220 KDa) --. -.- Figure 3.9: RA-togged Gcr3p does not interact with the CTD of the largest subunit of RNAP II Panel A: Whole-ce11 yeast extract containing HA-tagged Gcrîp was incubated with glutathione beads coupled to one of four ligands; GST protein alone, GST fused to 16 CTD repeats (GST-CTD), GST-fused to 16 CTD repeats and phosphorylated by HeLa extract (GST-CTDP)and GST fused to 15 mutant CTD repeats and phosphorylated (GST- ASP). Panel B: The ligands coupled to glutathione beads after SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue. Panel C: Beads were washed and eluted in high sait. Eluates were analysed either for the presence of HA-tagged Gcr3p by protein-blot anaiysis using anti-HA antibody or for the presence of guanylyl transferase by labelling eluates with [a-32~1-GTPand subsequent SDS-polyacrylamide gel electrophoresis. HA-tagged Gcr3p could be detected in as Little as
1.3% of the extract loaded ont0 the beads but was undetectable in any of the eluates. 20% of each eluate was loaded. Guanylyl transferase bound to [3*P]-GMP (gt-GMP*) was detected only in the eluate from the phosphorylated GST-CTD beads as expected. alutathione beads -GST or
1100 mM NaCl wash
guanylyl-transferase assay anti-HA protein-blot
eluates
Si%904 load GG ii'i'i'c, -eu 5sO O5
% loaded 1.3 2.5 5 10 20 20 20 20
load P.PG6a iO& ii'ii' 6% 0% e5 &
% loaded 0.15 0.3 0.6 6 6 6 6 gcr3-100 confers an in vitro splicing defect
GCR3 was identified originally as a gene which was required for efficient expression of glycolytic genes (Uemura and Jigami, 1992; Uemura et d., 1996). Later, it was shown to encode the Iargest subunit of a two-subunit complex that binds to the 5'-rnethylated- guanosine cap structure which is added to nascent RNA-polymerase II transcripts. This cap-binding complex (CBC) was also shown to play a role in effkient pre-mRNA splicing
(Lewis et al., 1996b; Lewis et al., 1996a) in yeast and mammalian systems and expon of U snRiVA to the cytoplasm (Gorlich et al., 1996; Izaurralde et al., 1995) in Xenopus. Yeast whole-cell extract that was irnmunodepleted of the CBC was found to be deficient in an in vitro splicing assay (Lewis et al., 1996b). Since gcr3-100 encodes a truncated version of Gcr3p (and confers an overail phenotype similar to that of a GCR3 gene disruption that removes the entire ORF), 1 investigated the possibility that gcr3-100 might confer a splicing defect as well. In vitro splicing activity in gcr3-100 extract was approximately 50% of that found in WT extract (Figure 3.10). This splicing defect (like the promoter-driven transcription defect) seems to be particular to the gcr3-100 mutation since 1 found no detectable splicing defect for my Agcr3 extracts (data not shown). The gcr3-100 splicing defect is similar to the 70-80% decrease in activity caused by immunodepleting cap-binding complex from a yeast extract (Lewis et al., 1996b). The fact that 1 found no splicing defect for my Agcr3 extracts suggests that this immunodepletion removes other splicing-related proteins from yeast extract besides just CBC (Lewis et al., 1996b). 1 added immunopurified CBC (see above) to gcr3-100 extracts, but saw no improvement in in vitro splicing activity (data not shown). This suggests that the gcr3-100 splicing defect is either an indirect one or WT CBC is unable to compte with the endogenous mutant gcr3- 100 protein. In conclusion, 1 have found that the gcr-3-100 mutation is syntheticaiiy lethal with the rpo21-30 mutation and that it confers an in vitro promoter-specific transcription defect and an in vitro splicing defect. These in vitro phenotypes seem to be specific to the gcr3- 100 mutation since they were not observed for a gcr3-delete strain. This raises the possibility that the synthetic-lethality observed between alleles of GCR3 and MO21 may also be specific to the gcr3-100 allele and that the failore of CBC to complement the in vitro defects of gcr3-100 is due to its inability to compete with an "in vitro dominant" mutant CBC. However, barring this possibility, the observations provided present no evidence for a direct role of Gcr3p in transcription. The observed defect in promoter-dependent transcription conferred by gcr3-100 may be indirect and the synthetic-lethality between rpo2 1-30 and gcr3-100 rnay be due to a combination of reduced transcription activity (rpo21-30)and pre-mRNA splicing ancüor export (gcr3-100). Alternatively, gcr3-100 may indirectly affect transcription by decreasing expression of a number of transcription-related genes that have introns (see Discussion). Figure 3.10: Extracts of gcr3-100 have reduced in vitro splicing activity.
Equal amounts of yeast whole-ce11 extracts from a WT strain (YF2094) and from the gcr3- 100 mutant (YF2349) were assayed for in vitro splicing activity using a pre-actin mRNA
(lane 1). The mobility of lariat-exon2, lariat, precursor and mature mRNAs are indicated to the left. Quantification was performed using Image-Quant software (Molecuiar Dynamics).
The ratio of mature to precursor is expressed as "%-spliced"at the bonom of each lane.
Discussion
A mutation in the zinc-binding domain of the largest subunit of RNAP II (rpo21-30) was used as a starting mutation to search for additional components of the transcriptional machinery in a synthetic-lethai screen. This method proved successful in the identification of a mutant allele of SRB5 that encodes a known subunit of the RNAP II holoenzyme. A second mutation was identified in the GCR3 gene. GCR3 was fmt identified as a gene involved in glycolytic gene expression (Uemura and Jigarni, 1992). The original mutant alleles (gcr3-1 and -2) of this gene were identified in a strain in which p-gaiactosidase activity from an ENOl-lad fusion reporter plasmid was decreased. This effect appeared to be mediated at the level of transcription because in vivo mRNA levels from the ElVOf-lacZ reporter consuuct were decreased in a Agcr3 strain. Additional evidence of a role for Gcr3p in transcription was published during the present study. The slow-growth phenotype of a gcr3 mutant can be suppressed by a top1 (topoisornerase 1) mutant and alieles of gcr3 were found that suppressed the temperature- sensitive growth defect of a Ahprl mutant (Uemura et al., 1996). While the molecular nature of these genetic interactions is still unknown, both Toplp and Hprlp potentially have roles in RIVAP II transcription. For instance, the ability of Toplp to relax DNA supercoils generated dunng transcription probably adds to the eficiency of transcription (Brill et al., 1987; Briil and Sternglanz, 1988; Gartenberg and Wang, 1992; Phoenix et al., 1997); interestingly, plasmid DNA isolated from a Agcr3 mutant is more negatively supercoiled than from a WT strain (Uemura et ai., 1996). Likewise, Hprlp has a demonstrated role in the passage of the transcription complex through some open reading frarnes (Chavez and Aguilera, 1997; Prado et ai., 1997); other suppressors of hprl mutants include RNAP 11 holoenzyme components including RPB2, TFIïBISUA7 (Fan et al., 1996; Fan and Klein, 1994), GALI 1, SIN4/RGRl (Piruat et al., 1997) and SRB2 (Pimat and Aguilera, 1996). Later, GCR3 was found to encode the largest subunit of the S'cap-binding complex (CBC)(Gorlich et ai., 1996). The CBC plays a number of potential roles in mRNA biogenesis, including splicing and 3'end cleavage (see Chapter 1). Taken together then, it is possible that the observed synthetic-lethality between rp21-30 and gcr3-100 is due to a reduced activity in any of a number of steps in mRNA biogenesis. Since, rpo2l-30 reduces overall in vitro RNAP II activity in extracts. 1 began with the possibility that Gcr3p plays an unsuspected role in promoter-directed transcription itself. No evidence for a direct involvement was found. Below, 1 discuss some of the possibilities that might explain the observed synthetic-lethality. These possibilities include: (1) a role in transcription initiation or elongation, (2) a role in splicing, (3) a role in 3'-processing and (4) a role in RNA export.
1. Gcr3p and transcription initiation and elongation The synthetic-iethality observed between gcr3-100 and rpo21-30 was not rescued at 30°C by other RP021 mutant alleies, suggesting that the interaction is probably not allele- specific. Somewhat inexplicably, rpo21-28 (C70S) was abIe to restore sectoring to the
P88 (gcr3-100) mutant at 23°C aithough it was unable to do the sarne for the P47 (srb5- 100) mutant It is possible that the C70S substitution is less severe than other ZBD substitutions; by itself, C70S confers only a slow-growth phenotype at 37°C. Alternatively, the C70S mutation may bypass the need for Gcr3p function that is required in the presence of other RP021 mutants. In any case, these results suggest that the identification of gcr3-100 does not reveal information relevant to the function of the zinc-binding domain. However, this did not rule out the possibility that Gcr3p is involved directly in the process of transcription, especially
boiven the other genetic interactions with TOP1 and HPR1 that had been noted before (see above). Consistent with this hypothesis, 1 found that gcd-100 extracts were 40% reduced in promoter-specific transcription activity. These differences were not due to variation between extracts; the differences were reproducible for two sets of extracts and other activities in these exrracts (promoter non-specific RNAPII and RNAP I+m transcription) were sirnilar in both WT and mutant extracts. The fact that no decrease was observed in
RNAP II non-promoter-specific activity does not rule out a Gcr3p-role in transcription since Srb5p also has no effect on promoter-non-specific transcription (Thompson et al., 1993). It seems unlikely that the gcr3- 100 promoter-specific transcription effect is direct because purified CBC had no stimulatory effect on transcription activity in gcr3-100 extracts (data not shown)- I aiso addressed the possibility that Gcr3p is a component of the RNAP II: holoenzyme. Given the fact that Gcr3p is a CBC subunit and that capping is thought to be co- transcriptional, this seemed a strong possibility. Nascent pre-mECNA that is capped could be bound immediately by the CBC,which in turn could direct the pre-mRNA substrate to further processing sites. The presence of CBC in the initiation cornplex could help explain how the gcr3-100 mutation was isolated as king synthetically lethal with rpo2 1-30 and how it is able to confer an in vitro transcription defect. However, no evidence was found for an association between CBC and RNAP II from CO-imrnunoprecipitationexperiments.
Later it was shown that the capping enzymes themselves were associated with RNAP II but only when the CTD is in the phosphorylated state (McCracken et al., 1997). This raised the possibility that CBC also associated with the CTD in its phosphorylated state during the elongation phase of transcription. 1 found no evidence that HA-tagged Gcr3p was associated with either phosphorylated or non-phosphorylated CTD, although 1 was able to repeat the observed interaction between guanylyl~ansferaseand the phosphory lated form of the CTD. In addition, Susan McCracken and David Bentley in collaboration with Iain Mattaj's group found no evidence for a CTD-CBC interaction (McCracken; personal communication). These results argue against a CBC interaction with the RNAP X holoenzyme but do not exclude the possibility. For instance, the possibility that such an interaction may require the presence of capped mRNA has not been addressed. Evidence has been reported to suggest that the capping enzymes may play a role in rnediating efficient transcription initiation. S-Adenosyl-L-homocysteine (AdoHcy) is a
produc t and an inhibitor of the methyltransferase capping enzyme (see Introduction, Figure 1.3 and (Mao et al., 1995)). AdoHcy is also able to inhibit the accumulation of RNAP II run-off transcripts in in vitro transcription assays using HeLa extract. It has no effect on RNAP ID transcription in extracts or on purified HeLa RNAP II (Jove and Manley, 1982) possibly because it is specific to RNAP II as part of a larger complex. AdoHcy probably
acts at some stage prior to elongation, since adding it after initiating transcription has no effect. Most of the transcripts that were made in the presence of AdoHcy were terminated at the S'-end with a non-methylated GpppA cap, indicating that methylation is not a pre- requisite for elongation. Although the precise mechanism of inhibition is unknown, it was suggested that AdoHcy rnight cause some conformational change in the initiation complex that could inhibit transcription (Jove and Manley, 1982). This hypothesis would predict that AdoHcy could inhibit transcription in a reconstituted system using purified factors if the capping enzymes were added; however, the AdoHcy effect on transcription has not been addressed since the original observation was made (Jove and Manley, 1982).
2. Gcr3p and spiicing The yeast CBC has a demonstrated role in splicing. Extracts irnmunodepleted of CBC (Gcr3p and Mud l3p) or made from a dmudl3 strain are deficient in the ability to form the splicing cornmitment complex when added to pre-mRNA (Colot et al., 1996; Lewis et al., 1996b). Addition of purified CBC or recombinant Mudl3p to these respective extracts is able to restore this defect (Colot et al., 1996; Lewis et al., 1996b). The same holds mefor splicing activity in these extracts although splicing is restored only partiaiiy by the addition of purified CBC and is restored poorly by the addition of recombinant Mudl3p to these extracts (see Chapter 1, section 3.2). 1 addressed the possibility that gcr3-100 extracts were deficient for splicing activity and found a 50% decrease in activity. 1 found no evidence that this effect was a direct one because purified CBC had no stimulatory effect on gcr3-100 splicing activity in vitro. One would also predict that gcr3-100 extracts are deficient in cornmitment-complex formation that could also be restored by the addition of purified CBC; however, this was not assayed for. No splicing defect was observed for Agcr3 extracts, suggesting that the gcr3-100 tmncation allele might interfere specifically with efficient in vitro splicing. This result also suggests that immunodepletion of CBC from extracts results in a splicing defect because other splicing factors are co-depleied; addition of purified CBC to these extracts is able to restore splicing only to 50% of \NT activity (Lewis et al., 1996b). A Agcr3 strain was constructed independently of this study and was shown to have no effect on in vitro splicing of actin pre-mRNA, although weak defects were observed for other introns (Puri Fortes and Iain Mattaj; personal communication). These differences between the gcr3-100 and Agcr3 alleles suggest that it may be possible to find extragenic suppressors of gcr3-100 that do not suppress the Agcr3 allele. Such suppressors may encode proteins that interact functionally with Gcr3p. The gcr3-100 splicing defect raises the possibility that the transcription defect observed for these same extracts might be related indirectly to underexpression of holoenzyme components whose open reading frames have introns. 1 searched the Yeast Protein Database and found four candidates that could fit this description; these included ANCl (a subunit of the SWUSNF complex and of transcription factor TFIIF), KIN28 (a CTD kinase), RPB6(a subunit of RNAP II) and SRB2 (a component of the RNAP II holoenzyme) (see Chapter 1, Table 1.1 and 1.2). Future experiments might involve comparing mRNA and protein expression from these open reading frarnes to confm this hypothesis. 1 hypothesized that the splicing defect of gcr3-100 coupled with the transcription defect of rpo21-30 may explain the synthetic-lethality between these two alleles. This hypothesis could be further tested by determining whether other mutations in splicing genes are syntheticaliy lethal with r@l-30. 3. Gcr3p and 3'-cleavage
Mamrnalian CBC has a demonstrated role in 3'-end cleavage of pre-rnRNAs (Chapter
1, Section 4.2). Cap does not appear to be required for pdyadenyiation of pre-rnRNA (see Chapter 1); however, it is less clear whether the presence of cap (and CBC) has any effect on 3'-end cleavage. This possibility could be addressed using the gcr3-100 and Agcr3
mutant extracts from this sîudy in an in vitro 3'-end cleavage assay (Butler et al., 1990). Such a defect would not explain the transcription defect observed in gcr3-100 extracts (these assays do not rely on 3'-end cleavage to determine the length of the transcript) but would be consistent with the identification of gcr3-100 as a mutation that is synthetically lethal with an RNAP II mutation. Furthemore, a 3'-cleavage defect could also help expIain the fact that plasmid DNA is hypernegatively supercoiled in a Agcr3 strain (Uemura et al., 1996). Since 3l-çleavage is required for termination of transcription and dissociation of RNAP II from template DNA, a decrease in 3'-end cleavage activity would cause accumulation of non-terminated mRNA-DNA-RNAP II ternary complexes. Such complexes have been demonstrated to enhance the formation of R-loop structures
(extended RNA-DNA hybrids) in bacterial systems, which in turn increase the negative
superhelicity of plasrnid DNA (Phoenix et al., 1997).
4. Gcr3p and export The importin-a@ complex of yeast and higher eukaryotes is able to bind proteins that have a nuclear localization signal (NLS) and mediate their transport into the nucleus. The largest subunit of the yeast and Xenopus cap-binding complex (CBC) has a NLS. About one third of yeast CBC is associated with one third of the importin-a (Gorlich et al., 1996). The CBC/importin-a subcomplex is able to bind to 5'-m7~ppp~capped RNAs. In Xenopus, this complex is involved in exporting U snRNAs made by RNAP II to the cytoplasm and in re-importing CBC back into the nucleus (see Chapter 1, Section 6.1). It is likely that this is also the case in yeast, although the export of U snRNAs have never been demonstrated in this system for technicd reasons (Gorlich et ai., 1996). U snRNAs in metazoans are exported to the cytoplasm where they are tri-methylated, assembled into snRNPs with other proteins and then re-imported into the nucleus where they splice pre- mRYAs (Stutz and Rosbash, 1998). This potential involvement of Gcr3p in U snRNA export is probably not the explanation for the splicing defect seen in gcr3-100 extracts, for the following two reasons. First, a splicing defect cm be conferred in yeast extract by immunoprecipitating CBC and then partially corrected by adding recombinant CBC back to the extract (Lewis et al., 1996b). Second, extracts made from a gcr3-deletion strain have no visible splicing defect (this study). 1 conclude from this that the in viîro splicing defect conferred by gcr3-100 is not due to decreased concentrations of nuclear snRNPs. RNAP II mRNA transcripts are also capped but it does not appear as though the CBChmportin-a system is required for export of these RNAs; a Agcr3 mutant does not accumulate nuclear poiy(A) RNA (Gorlich et al.. 1996) and SRPI has no demonstrated effect on mRNA export (Loeb et al., 1995). This suggests that the synthetic-lethality observed between rpo21-30 and gcr3-100 is not related to a potentiai role of Gcr3p in mRNA export.
An allele of yeast importin-a (SRPI- I) was identified as a dominant suppressor of a mutation in the zinc-binding domain of the largest subunit of RNAP 1 (Yano et ai., 1992). This substitution mutant (rpal-5; HSOY) is analogous to the RNAP II mutation used for the synthetic-lethal screen described in this chapter (rpo21-30; H80Y). However. I showed that SRPl-I was unable to suppress the temperature-sensitive phenotype of rpo21-30 (data not shown) indicating that either the SRPl-l allele or srpl itself was irrelevant to the function of RNAP II. The mechanism by which SRPI-l suppresses rpal-5 is unclear. Other mutations in srpl were shown to confer pleiotropic effects including instability of the nucleolar sub-structure (in which RNAP 1 functions)(Yano et al., 1994). It is possible that the ability of SRPI-1 to suppress the temperature-sensitivity of rpal-5 is related to its ability to mediate import of nuclear proteins related to RNAP 1 function, although this has never been demonstrated. It also seems likely îhat srpl-1 does not suppress rpo21-30 because it alters the import of proteins that are related specifically to RNAP 1 function and not RNAP II function. The identification of SRPI-I as an allele-specific, dominant suppressor of rpaf -5 (H80Y) (Yano et al., 1992) is not likely related to the interaction of Srplp with Gcr3p (of the cap-binding complex) even though a mutant allele of GCR3 was identified (in this work) as king syntheticdy lethal with @l-30 (HSOY). RNAP 1 transcripts (unlike
RNAP II transcripts) are not capped and so their biogenesis is unlikely to be influenced directly by the SrplpKBC complex. This hypothesis would predict that gcr3-IO0 is not synthetically lethal with pal-5. Srp lp (yeast importin-a) is required for nuclear import of proteins that have a nuclear localization signal (NLS). Gcr3p has a putative N-terminal
NLS and is only one of many proteins that are irnported to the nucleus via yeast importin. References
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Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993).
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U S A 91, 6880-4. Chapter N Characterization of a mutant srbS splicing defect and the effect of an
RNAP 11 carboxyl-terminal domain peptide on yeast in vitro splicing.
Sequence analysis of srb5-100 was done at the HSC-Phannacia Biotechnology center. Abstract Given that a number of splicing-related factors cm interact with the mammalian carboxyl-terminal domain (CTD)of the largest subunit of RNAP II (section 3.2), it seemed plausible that the original genetic screen for suppresors of CTD truncation mutants may have identified components of the splicing machinery (Thompson et al, 1993). One of the suppressors identified in this screen was an allele of SRBS. This chapter describes an attempt to determine if Srb5p also plays a role in splicing. Extracts of the mutant SRBS strain were deficient in splicing activity- However, recombinant WT SrMp was unable to restore splicing activity to mutant extracts suggesting that the effect was indirect. In addition, a CTD peptide was unable to inhibit splicing in yeast extract even though it is able to inhibit splicing in HeLa extract (Yuryev et al, 1996). This suggests that the CTD peptide and/or yeast extract behave differently fkom CTD peptide in HeLa extracts or, aiternatively, that splicing components may not associate with the yeast CTD as in the mamrnalian system. I hypothesize that the mutant SRBS splicing defect is an indirect one, possibly mediated by the effect of the SRBS mutant allele on the gene expression of one or more splicing factors. In support of this indirect hypothesis, 1 show that extracts made from Rpo2lp- ZBD mutants also were reduced in splicing activity. This raises the possibility that splicing activity is generally sensitive to defects in components of the transcriptional rnachinery. Introduction One of the genes identified in the synthetic-lethal screen with rpo2ï-30 was an allele of SRB5 (see Chapter 3). This gene was identified originally as a dominant, aiiele-specific suppressor of a truncation of the C-terminal domain of Rpo2lp (suppressor of ENA polymerase (Thompson et ai., 1993). SrbSp and Srb2p are required in vitro for efficient, promoter-specific transcription and template cornmitment by RNA poiymerase II, but not for non-specific transcription in vitro (Thompson et al., 1993). In addition, SrbSp, dong with several other proteins identified in the same screen, were shown to be a part of
the RNAP II holoenzyme and to be associated physically with the C-terminai domain (CTD) (Kim et al., 1994; Koleske and Young, 1994). The identification of SM5 in my
synthetic-lethal screen lent credence to the rationale of the screen and its ability to identiw other components of the RNAP Ii holoenzyrne. This chapter describes an analysis of the srb5-100 mutation. The data demonstrate that this aiieIe directly confers an in vitro, promoter-specific transcription defect. In addition, 1 address the possibility that Srb5p plays a role in splicing. The study of the effect of srb5-100 on splicing was prompted by two earlier findings. First, SRB5 was isolated originally in a suppressor screen for mutations that could suppress the growth phenotypes associated with a truncated RNAP II CTD (Thompson et al, 1993). Given the association between marnmalian splicing and the CTD (see Chapter 1,
Section 3. l), it is conceivable that this screen could have identified pre-mRNA processing factors that were associated with the CïD . Second, the in vitro splicing defect of gcr3-100 extracts could explain why this allele was synthetically lethaî with rpo2l-30 (Chapter 3). These considerations raised the possibility that gcr3-100 and srb5-100 were identified in my synthetic-lethal screen because they both conferred splicing defects. For these reasons, extracts of the srb5-100 mutant were made and assayed for splicing activity. They were found to be significantly deficient in splicing activity with respect to WT extracts. However, additional experiments indicated that this effect is likely an indirect one. The RNAP II largest subunit carboxyl-terminal domain (C'ïD) is another transcription related factor that has ken implicated in splicing. Recently, two groups have demonstrated that mammalian splicing activity cm be inhibited by the RNAP II CTD, either in vivo or in vitro (Kim et ai., 1997; Yuryev et al., 1996). This effect is thought to be mediated by the ability of the added CID to dissociate splicing-related factors from the endogenous CTD thus interfering with their coordination in the splicing reaction. While SR-related proteins from HeLa ce11 extract can bind to the CID, no evidence has yet been found for the binding of splicing-related factors to the yeast RNAP II CTD. However, if splicing and transcription are coordinated in yeast, as they are thought to be in mammalian systems, then one would expect that the CTD peptide would also inhibit splicing in yeast extracts. 1 repeated an in vitro ClD inhibition experiment in yeast extracts and found no inhibition of splicing. This suggests that the CTD and/or yeast extract behave differently than CTD in HeLa extracts or, alternatively, that splicing components may not associate with the yeast
CTD as in the mammalian system. In considering how the SRBS splicing defect could be explained, 1 hypothesized that it rnight be an indirect one, possibly mediated by the effect of the SRBS mutant allele on the gene expression of one or more splicing factors. in support of this indirect hypothesis, 1 showed that extracts made from Rpo2lp-ZBD mutants also were reduced in splicing activity. This raises the possibility that splicing activity is generally sensitive to defects in components of the transcriptionai machines). Methods and Materials Yeast strains and media Yeast strains used in this study are listed in Table 4.1 and their construction is described in Chapters 2 and 3. Media and yeast genetic methods are described in Chapter 3.
Identification of the mutation in sr65-100 The mutant srb5-100 and corresponding WT sequences were ampiified from the chromosome of yeast strains YF2105 and YF2070 respectively using oligos ID055 (5'- GGGGAAGCTTGATCTTCAGTATCCTCGCGG-3') and ID056 (5'- GGGGGGATCCGATCAAGAAGTTGTTGCTGG-3'). These primers ampli@ a 1.9 Kb fragment from the chromosome and add Hi- (IDOSS) and BamHI (ID056) restriction sites to the ends. Reactions were in 100 fl containing dATP, dGTP, dCTP and dTTP (0.2 rnM each; BRL), 0.5 pg of each oligo, 1 pl of yeast (treated with Zymolase as described in (Ling et al., 1995)), 3.5 U Taq/PWO Kigh Fidelity polymerase (Boehringer Mannheim), in a buffer supplied by the manufacturer with 15 mM MgC12. The reaction conditions were as follows: 95°C for 5 min., 10 cycles of (95°C for 1 min.; 45°C for 1 min.; 72°C for 2 min.), 20 cycles of (95°C for 1 min.; 55°C for 1 min.; 72°C for 2 min.) and 72°C for 10 min. The reactions were repeated using four separate colonies for each strain. Electrophoresis revealed a single 1.9 Kb producc. Sequence information was obtained on both strands for the mutant and WT PCR products (HSC Biotechnology Service Center) between nucleotides -167 (where the A of the initiating ATG is +1) and +935 (the ORF ends at nt +924). Table 4.1: Yeast strains used in this study Strain relevant eenotvw MATa caril-100 his3-1 l,l5 Ia42-3,112 Ltpl-1 trrn3-1 de2-1 ssûl -d (aka YF554) W3031a with rpoZI::HIS3 pYP1577(pGALIO-RP021 URA3 CEN ARS) W3031a with rpo21 ::HIS3 pY FI550 (rpo21-3OTRPl CEN ARS) W3031a with rpo21::HlS3 pYF1556 (rpoZ1-36TRP1 CEN ARS) W303la with rpo21 ::HIS3 pYF1557 (rpo21-37 TRPl CEN ARS) W3031a with rpo2l::HfS3 pYF1513 (RPOZI TRPl CEN ARS) MATa ml-100 his3-11,15 îu2-3,Z 12 trpl-1 um3-1 aàe2-1 ade3::HisG 41-d GAL psi* (aka YF2035 from Fatima Cvrckova; derivative of W303-la) W303-lb with rpoZ1::ADEZ pYFl577(pGALlO-RPOZl URA3 CEN ARS) MATa rpoZ1-30(H80Y) cm1-100 his3-ll,lS leuZ-jJ 12 trpl-1 ura3-1 adc2-1 dl-d (derived from Y Pl733) MATa RPO21 cd-100 his3-11,15 leu2-3,112 trpl-1 ura3-1 ade2-1 ssdl-d (derived from YF1733) MATa rpo21-3qH80Y) can 1-100 his3-11 ,lS leu2-3, Il2 trpl-1 ura3-1 ade2-1 ssdl d rde3::HisC (deriveâ from Y FîO66) MATa rpo21-30(H80Y) canl-100 his3-11 ,15 lm2-3, 112 trpl-2 ura3-1 ade2-1 ssdl -d d3::HisG (pYF1465(RP021 URA3 ADE3 2p)) derived from YF2074 srb5-100 UV-induced mutant of YF2079 (aka P47) MATala K2346 x Y F2O74 MATa RP021 ml-100 his3-11 ,15 le&-3, 112 trpl-1 ura3-1 ade2-1 ssdl-d nde3::HisG YEpl3 (LEUî)(spore product of Y FîO93) MATalaYF2105 x YF2094 MAT ? RP021 sib5-100 W-1 ade3::HirC (spore product of YF2341; aka Tl-1) YF2347 MAT ? RPO21 sh5-100 ade2-1 ade3::HisG (spore product of YF2341; aka T7-4) Purification of recombinant SrbZp and SrbSp Plasrnids expressing recombinant SrbZp (pTK-27)(Koleske et al., 1992) and SrbSp (pCT98)(Thompson et al., 1993) in the E. coli BL2 l(DE3)(Studier and Moffatt, 1986) were a gift from Rick Young (see pYF1785 and pYF1786). Protein was expressed and purified from inclusion bodies as described in (Koleske et al., 1992) . E. coli expressing recombinant Srb2p and Srb5p under the control of a T7 RNA polymerase promoter were grown in 2 L of LB medium each supplernented with 200 pg/ml of ampicillin at 37°C to an OD600 of 1.O. Expression of T7 RNA polymerase was induced by the addition of isopropyl $-galactoside to 0.5 mM. Cultures were incubated for an additional 3 hr, harvested by centrifugation and resuspended in 40 ml of KPN buffer (20 mM potassium phosphate (pH 7.3, 150 mM NaCl). Cells were lysed by mild sonication on ice (4 min. at setting 5; 50% duty cycle). The bacterial lysate was centnfuged at 12 Krpm in a Sorvall SS-34 rotor and the pellet was resuspended in 4 volumes of Laernmli buffer (60 rnM Tris-HC1 (pH 6.8), 10% glycerol, 2% SDS, 144 mM PME) and boiled for 10 min. Debris was pelleted by centrifugation at 12 Krpm for 10 min. in a Sorvall SS-34 rotor. The recombinant protein lysate was then mixed with bromophenol blue to .ûû1%, heated to 65 OC for 10 min. and loaded into 10 wells each on 12.5% SDS-polyacrylamide gels with 3 mm spacers ( 35 pl of SRBS and 70pl of SRBS per well determined empirically so as to not overload the gel). Electrophoresis was at 200 v for 3.5 hours. One lane of each gel was stained with Coomassie-blue for 20 min. (with 3 x 2 min. in the microwave at medium setting) and then destained in a sirnilar manner. The recombinant protein was excised from the remaining lanes according to the mobility in the fust lane and a molecular weight marker (Srb2p at 23 kDa and SrbSp at 34 kDa). Each gel strip was then minced, passed through a 10 ml syringe (with no needle), combined with 2 volumes of IX SDS-
PAGE running buffer (25 mM Tris, 192 rnM glycine, and 0.1 % SDS (pH 8.3)) and turned end-over end in a 50 ml tube overnight at 23OC. The gel was removed by filtration through
Whatmann paper and protein was precipitated by adding 4 volumes of acetone (at -20°C) and incubating on a dry-ice/ethanol bath for 1 hr. Protein was pelleted by centrifugation at 10 Krpm for 10 min. in a SS-34 Sorvall rotor. Each protein pellet was resuspended in 3 ml of HKA buffer (20 mM HEPES (pH 7.5). 150 mM potassium acetate) containing 6 M
urea and dialyzed against 2 L of HKA buffer with several changes each time with successively reduced concentrations of urea: 6 M urea for 3 hr., 3 M urea for 4 hr., 1.5 M
urea for 4 hr., 0.75 M urea for 9 hr and finally without urea for 4 hr. Lastly, the proteins were dialyzed against IL of TDB (20 mM HEPES pH 7.5,20% glycerol, 10 mM MgS04,
10 mM EGTA, 5 rnM DTT, 1 mM PMSF) for 6 hr with two changes of buffer and then stored at -70°C-
In vitro promoter-specific transcription and splicing assays for yeast The preparation of whole-cell extracts and in vitro assays for transcription and splicing are described in Chapters 2 and 3.
Assay of in vitra splicing activity in HeLa cell nuclear extract Capped and 32~-labelledpre-mRNA was synthesized from plasrnid pPIP85 DNA ((Moore and Sharp, 1992) obtained from Andrew McMiilan; see pYF1758) using T7 RNA polymerase as follows. pPIP8S plasmid DNA (2 pg) digested with HindIII was transcribed by T7 RNA polymerase for 1.5 hrs at 23°C in 18.5 pl containing 30 U T7 RNA polymerase (Pharmacia), 40 mhf Tris-HC1 (pH 7.5),6 mM MgC12, 10 mM NaCl, 2 rnM sperrnidine, 10 mM DTT, 1 m.each of ATP, GTP, CTP, 50 pM UTP (Pharmacia), 0.9 mM rn7~(5')~~~(5')~(Pharmacia), 50 pCi [GC-~~PI-UTP(3000 Ci/mmol; Mandel), 16 U RNasin (Promega). DNase 1 (30 U, Nase-free; Boehringer Mannheim) was added and incubated at 37°C for 20 min. to remove template DNA. The reaction was extracted with phenol/chloroform/isoarnyl alcohol (2524: 1), precipitated twice with ethanol, washed in 70% ethanol and fmally resuspended in 25 pl distilled water and stored at -20°C. In vitro HeLa ce11 splicing reactions were carried out in a total volume of 15 pl containing 2 m.MgCl2, 1.5 mM ATP, 5 rnM creatine phosphate (Sigma), 5 rnM DTT, 40
units RNasin (Promega), 5 pl of nuclear extract (10 mglml; gift from Ben Blencowe prepared as described in (Dignarn et al., 1983)) and 0.5 pl of transcript from the labelling reaction described above (approximately 18 Kcpm as merisured by a table-top BIOSCAN counter). Reactions were started by adding extract and were incubated for up to 2 hours at
30°C. Reactions were stopped by adding 5 pl of 4X stop mix (80 mM Tris-HCI (pH 8.0),
80 mM EDTA, 2% SDS, 2.4 mghl proteinase K (> 20 unitdmg; Gibco-BRL), and 0.72 mg/d E. coli tRNA (Boehringer Mannheim). Reactions were incubated for 40 min. at 65°C to digest protein. Proteinase K buffer (300 pl containing 100 mM NaCI, 10 mM Tris-HC1 (pH 7.6), 1 mM EDTA, and 0.5% SDS) was added dong with 10 pg of glycogen (Boehringer Mannheim). The reaction products were extracted with phenol: chloroform: isoamyi alcohol(25:24: l), ethanol-precipitated and washed with 70% ethanol, dned in a speed-vacuum for < 5 min. and were fially resuspended in IO pl of loading dye (90 rnM Tris-borate, 1 rnM EDTA, 90% (w/v) formamide, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol). Half of the reaction products were heated to 65°C for 10 min. before loading on a pre-run, 15% polyacrylamide (0.2% bisacrylamide) gel with 7M urea (0.8 mm spacers). Electrophoresis was in 1X TBE (90 mM Tris-borate, 1 mM EDTA) for 3 hours at 500 volts (until the xylene cyanol band was at the bottom of the gel). The gel was transferred to a piece of acetate, sealed in plastic and exposed to film (BIOMAX-MR; Kodak) or a MoIecular Dynamics phosphor screen for 6-24 hours. Preparation of CTD peptide The 56 residue peptide consisting of 8 repeats of the CTD heptamer (H2N- (S~P~T~S~P~S~Y1)8-~~~H)was prepared as described in (Cagas and Corden, 1995) by Bioworld (Dublin, Ohio) using standard methods employing t-Boc amino acids. The peptide had a calculated rnolecular weight of 5,775.4 and a molar extinction coefficient of 10240 (M)A~~~-~(~Y%)such that an absorbance of 1.0 at 280 nm corresponds to 0.56 mg/rnl. CTD peptide was resuspended in 5 rnM HEPES (pH 7.5) to a concentration of 6 mg/d and stored at -70°C. Results
Identification of the mutation in srb5-100 and an associated in vitro promoter-specific transcription defect DNA sequence containing the SRBS ORF was amplified from the chromosome of the srb5-100 mutant that had ken isolated in the synthetic-lethal screen, and from the WT parental strain. Sequence analysis of both strands revealed a single thymidine insertion (Figure 4.1) foLiowing nuckotide 24 in the mutant DNA. The resulting frameshift mutation encodes a tnincated peptide of 12 amino acids. The sr6.5-100 mutation essentiaiiy removes most of the ORF and confers a temperature- sensitive phenotype similar to the SRBS gene disruption strain (Thompson et al., 1993).
Therefore, it was predicted that the srb5-100 mutant would also confer a severe defect in in vitro promoter-specific transcription activity like the SM35 disruption strain characterized by Thompson et al. (1993). This hypothesis was tested using extracts made from the srb5-
100 mutant. The srb5-100 strain isolated in the synthetic-lethal screen (YF2105) was back- crossed to a strain carrying WT alleles of both RPOZl and SRB5 (YF2094). Spore products carrying only the srbS-100 mutant allele were selected (see YF2344 and 2347).
The temperature-sensitivity of these spore products at 37°C was complemented by a plasmid carrying a WT copy of SRBS (pYF1573) but not by plasrnid alone (data not shown). Two independent mutant extracts as well as two WT controls (YF2094) were prepared for the experiments Listed below. Equal amounts of WT and srbS-100 extract protein were compared for in vitro, promoter-specific transcription activity (Figure 4.2 A). Quantification by phosphor image analysis revealed that activity present in mutant extract was 10-15% of that present in WT extract. This defect was specific to promoter-initiated transcription since RNA polymerase II non-specific activity was pater than 120% of that found in a WT extract (Figure 4.2 B). Furthemore, this defect was specific to RNA polymerase II since combined RNA MVQQLSLFWIYWZ srb5-100 CRF atg gtt cag caa cta agc ctt ttt tgg atc tac tgg tga ... *
S.WS ORF atg gtt cag caa cta agc ctt ttt gga tct att ggt gat gac ggc tac ... MVQQLSLFGSIGDDGY ...
Figure 4.1: The srb5-100 mutation is a single t insertion after nucleotide 24 (indiateci by a (*); where the 'a' of the initiating a$ is +1. The resulting frameshift -tes a truncated peptide of 12 amino acids (Z=stop codon). The wild-type SRB5 sequence is shown beiow. Figure 4.2: Extracts of srb5-100 are deficient in promoter-specific transcription activity Panel A: Increasing arnounts of whole-ce11 extract were assayed for promoter-specific transcription activity using pGAL4CG- as template. WT extract (40,80 and 120 pg from YF2094; lanes 1-3) was compared to a srb5-100 mutant strain extract (40,80 and 120 pg from YF2344; lanes 4-6). Transcription products were detected using a phosphor screen
(Molecular Dynamics). Quantification of the data (see text) was with Image-Quant software (Molecular Dynamics). These assays were repeated for two independent sets of extrac ts. Panel B: Whole-ce11 extracts (20 pg/assay) from the same strains were assayed for non- specific transcription activity using calf-thymus DNA as template. Al1 reactions were at
23°C for 20 min. and were repeated for two sets of extracts. Combined activities from RNA polymerase 1 and III were measured in the presence of a-amanitin and were subtracted from total RNA polymerase activity (no a-amanitin present) to obtain RNA polymerase II activity. The bars for each measurement represent the average of three trials where the standard deviation was less than 5%. For each extract, the RNA polymerase II activity is represented by a black bar (measured as a percentage of total activity in the extract) and by a stippled bar (measured as a percentage of RNA polymerase II activity present in an equivalent amount of WT extract). The combined activities of RNA polymerases I and III are shown by the grey bar expressed as a percentage of 1 and III ac tivity in WT extract. These assays were repeated for two independent sets of extracts. GAL4p binding site CYC1-TATA ?+
RNAP II with respect to total (%) RNAP II with respect to WT (%) RNAP I and III with respect Io WT (%) polymerase I and IIX activity were at least 80% of that present in a WT extract. These data confm that the identified dele has an in vitro phenotype very similar to that of drr65.
These data suggest a reasonable hypothesis explaining how the srb5-100 allele was isolated as synthetically lethal with a mutant allele of RPO21;rpo21-30 decreases activity of RNAP II in both promoter-dependent and independent transcription and is intolerable in the presence of a second promoter-specific defect conferred by the srb5-100 mutation.
Extracts of srb5-100 are deficient in splicing activity 1 wanted to determine if srb5-100 also conferred a splicing defect because previous studies suggested that this was a possibility (see htroduction of this Chapter). The extracts that were assayed for transcription activity were also assayed for actin pre-mRNA splicing activity. The srb5-100extract had a significant defect (10-15% of WT activity; see Figure
4.3). This is consistent with the hypothesis proposed above that the original screen used to identie SRB proteins may have identified proteins interacting with the CTD and required for efficient splicing. On the other hand, a mutation in SRBS might have an indirect effect on the ability of an extract to carry out splicing. A direct role of SrbSp in splicing was tested using recombinant SrbSp and Srb2p from E. coli. SrbZp was aiso added back because Srb2p levels were obsewed previously to decrease in a Asrb5 strain (Thompson et al., 1993). .These proteins were tested for function by adding them back (individually or in combination) to a mutant sr65-100 transcription reaction. As expected, SrbSp was abIe partially to restore promoter-dependent transcription activity (Figure 4.4; upper panel). This effect was increased in the presence of Srb2p. However, these same proteins were unable to restore splicing activity in a sr6.5-100 extract (Figure 4.4; lower panel). This result is not consistent with a direct role for Srb5p in splicing. On the other hand, it does not rule out this possibility since there are a number of reasons why this expriment might not detect restorative ability. First, the recombinant protein made in E. coli may not be folded or rnodified properly such that the relevant activity is not present or insuflicient to Figure 4.3: Extracts of srb5-100 are deficient in splicing activity. Whole-ce11 extracts from a WT strain (80 pg from YF2094; lanes 2-4) and a srb5-100
mutant strain (80 pg from YM347; lanes 5-7) were assayed for in vitro splicing activity of a capped, labelled pre-actin mRNA transcript (lane 1). The mobility of lariat-exon-2, lariat, precursor mRNA and mature product are indicated to the left. The ratio of mature to precursor (expressed as a percentage) is listed at the bottom of each lane. Precursor and spliced products were detected using a phosphor screen (Molecular Dynamics).
Quantification of the data (see text) was with Image-Quant software (Molecuiar Dynamics).
This assay was repeated for two sets of independent extracts.
Figure 4.4: Recombinant SrbZp and Srb5p do not restore the splicing activity of srb5-200 extracts Whole-ce11 extracts from a WT strain ( 80 pg from YF2094; lanes 1 to 4) and the srb5-100 mutant strain (80 pg from YF2344; lanes 5 to 8) were assayed for in virro prornoter-specific transcription activity (Top panel) or pre-actin mRNA splicing activity (Bottom panel). Assays were done with extract alone (lanes 1 and 5) or in the presence of purified Srb2p andor SrbSp. (250 ng each; Ianes 2 to 4 and 6 to 8).
No fwther restoration of the transcription defect was seen by adding back four times as much SrbS and Srb2 protein (data not shown). Quantification of autoradiograms was with NIH Image software. Numbers below lanes in the top panel indicate promoter-specific transcription product in each lane expressed as a percentage of WT (lane 1). Numbers below the bottom panel represent splicing activity expressed as a percentage of WT (lane
1 >- % of WT activiîy
% of Wf activity 100 103 99 87 51 47 43 39 iane 12345678 restore activity. Second, the absence of restored activity may be a function of the extract.
For example, SrbS protein added to sr65400 extract may be unable to assemble into a complex that is functional in the splicing assay. In support of this, I note that Srb5p and Srb2p are unable completely to restore the transcription activity of srb5-100 extracts,
suggesting that their true bnction is not replaced entirely by the recombinant proteins. In addition, components of the transcnption/splicing rnachinery may be irreversibly altered or darnaged in the srb.5-100 extract; this idea is supported by the appearance of what appear to
be Rpo2lp degradation products in the srb5-100 extract (see Figure 3.3 in Chapter 3).
Third, restored activity may be insufficient to be observable; since restored transcription activity is not complete (Figure 4.4 lane 8; top panel) it is questionable whether the effect of recombinant Srb5/2p would even be observable in the splicing assay especially if the recombinant protein does not restore activity to the same degree in both assays. These possibilities are difficult to rule out and the possibility remains that the srb5-100 splicing defect is an indirect one; srb.5-100 may alter the expression of gene products required for in vitro splicing activity. 1 present reasons for this last hypothesis in the Discussion.
CTD peptide and in vitro splicing activity of yeast extracts At the time that these experirnents were king carried out, Chabot et al. (1995) and Yuryev et al. (1996) showed that splicing in HeLa extracts could be inhibited by a monoclonal antibody directed against the CTD of the largest subunit of RNA polymerase II or by a synthetic, CTD-!ike peptide (8 repeats of YSPTSPS) (Yuryev et al., 1996). The basis of this inhibition was thought to involve a set of SR-like proteins that are found in association with the CTD in HeLa cells (Yuryev et al., 1996). SR proteins function in mammalian pre-mRNA splicing (Fu, 1995), although there is no evidence to suggest that SR-like proteins are involved in yeast pre-mRNA splicing. The sequence of the yeast CTD repeat consensus is identical to the mammalian one; thus it seemed reasonable to ask whether the CTD could inhibit splicing in yeast extracts as it does in HeLa extracts (Yuqev et al., 1996).
Although the CTD peptide was able to inhibit HeLa splicing (Figure 43, it showed no such activity in the yeast extract (Figure 4.6). This result indicates that yeast splicing cannot be inhibited by CTD peptide as it is in HeLa extract. Splicing and transcription factors may not be coupled together in yeast cells as they are in the marnmalian system.
Altematively, the CTD peptide may not be folded properly or phosphorylated in yeast extract to the same extent as it is in HeLa extract.
The above results do not support a direct role of Srb5p or the CTD in yeast splicing. Two classes of mechanism could explain an indirect Srb5p effect. First, decreased transcriptional activity might reduce the expression of a subset of genes that are required for splicing. Second, decreased transcriptional activity may generally down-regulate splicing activity in yeast whole-cell extracts (see Discussion). In either case, a hypothesized indirect effect of transcription activity on splicing activity would predict that other mutations in RNA polymerase II and related transcription factors might also confer splicing defects. To test this hypothesis, 1 prepared extracts from several of my original ZBD mutants grown at the permissive temperature. Each of these mutant extracts had splicing defects although smaller than that of srb5-100 (Figure 4.7). Quantification revealed that each of the three RP021 zinc-binding domain mutants had only 50% of the splicing activity present in the same arnount of protein from a WT extract. Purified RNAP II core enzyme was unable to reverse this defect (data not shown). This provides evidence (but does not prove) that the observed effect of mutant rpoZl and srb5-100 on splicing could be an indirect one. In addition, this result raises the possibility that both srb5-100 and gcr3-100 may have ken identified in the synthetic-lethal screen due to their effect on splicing. Additional experiments that would test this hypothesis and distinguish between the two mechanisms leading to a loss in splicing activity are proposed below. Figure 4.5: A CTD peptide inhibits splicing activity of HeLa extracts.
HeLa ce11 nuciear extract was assayed for the ability to splice pre-mRNA in the presence of 1.5,3, 6, 12 and 24 pg of 0 peptide that was either added directly to the reaction (lanes 5-9) or pre-incubated with HeLa extract for 15 min. at 30°C previous to the splicing reaction (lanes 10- 14). Lane 1 shows unspliced precursor mRNA. A time course of the reaction is shown in lanes 2-4 where buffer alone without CTD peptide has been added as a control. Al1 other reactions were for 60 min. The mobility of lariat-exon-2, precursor mRNA, lariat and mature product are indicated to the left. Quantification was by densitometry of scanned autoradiograms using NtH Image software and revealed that 6 pg of CTD peptide was sufficient to reduce spiicing to 60% of that obsewed at 60 min. with no peptide added. Spliced product was not detectable in the presence of 24 pg of CTD peptide. min. CTD pre-added CTD Figure 4.6: Addition of CTD peptide to yeast whole-cell extract has no effect on in vitro splicing activity. Yeast, whole-ce11 extract (80 pg) was assayed for the ability to splice actin pre-mRNA in the presence of 4 p1 of 5 mM HEPES (pH 7.5) containing 0, 1.5, 3, 6, 12 or 24 pg of CIB peptide that was added directly to the reaction (lanes 2-7) or that was pre-incubated with the extract for 10 min. at 23°C before adding substrate pre-mRNA (lanes 8-13). Unspliced mRNA is shown in lane 1. Al1 reactions were for 15 min. The mobility of lariat-exon-2, lariat, precursor and mature product are indicated to the left. Quantification was by densitometry of scanned autoradiograms using NM Image software and revealed no significant difference between splicing activity in the presence or absence of CTD peptide. CTD pre-added CTD fO-0- Figure 4.7: Extracts of RP0214BD mutants have reduced splicing activity Equal amounts of yeast whole-ce11 extracts (80 pg) fkom a WT (YFî155) strain and from RP021 zinc-binding domain mutants M 1 (YF2 15 l), M2 (YF2 152) and H80Y (YF2145) were assayed for in vitro splicing activity. The mobility of lariat-exon-2, lariat, pre-cursor mRNA and mature product are indicated to the left. Lariat was not resolved from precursor on this gel. Splicing products and intermediates were detected using a phosphor screen
(Molecular Dynamics). Quantification of the data was with Image-Quant software (Molecular Dynarnics). The ratio of mature to precursor (expressed as a percentage) is listed at the bottom of each lane. These results were repeated twice for two different sets of extracts with similar results. Addition of purified core RNA polymerase iI (8 to 800 ng per splicing assay) was unable to restore WT-iike splicing (data not shown). % spliced 18206985795710 Discussion The srb.5- 100 mutant allele encodes a one-nucleotide insertion that shifts the open reading frame which results in a deletion of the entire rernaining open reading frarne. Consistent with this, extracts of srb5-100 have a defect in promoter-directed transcription that is sirnilar to that conferred by the Ar65 ailele (Thompson et al., 1993). Non-specific transcription activity in both srbS-100 and dsrb5 extracts is unaffected since Srb5p is a component of the RNAP II holoenzyme and is not required for this activity. The promoter- specific transcription defect coupled with the generai decrease in RNAP II activity conferred by rpo21-30 is sufficient to explain why the mutant allele was identified in the synthetic-lethal screen. srb5-100 also conferred an in vitro splicing defect. This was unexpected since other activities in srb5-100 extracts were sirnilar to WT (RNAP U and I+m non-specific activity) and since SrbSp had not previously been implicated in splicing. 1 pursued the possibility that this splicing defect was caused directly by the lack of functional SrbSp in these extracts by adding back recombinant SrbSp and Srb2p made in E. coli. Although these proteins were able partially to restore the transcription defect of srb5-100 extracts, they had no observable effect on the splicing defect. This suggests that the splicing defect of srb5- 100 is indirect and is not related to a coupling between splicing and the CTD of the RNAP II holoenzyme. 1 cannot rule out the possibility that recombinant SrbSp is unable to reassemble into a larger complex that is important for splicing activity in srb5-100 extracts. However, 1 have obtained a strain of yeast from Rick Young that carries an epitope-tagged version of SrbSp. Imrnuno-depletion of SrbSp from extracts of this strain will be used to determine whether in vitro splicing activity is affected by the removai of SrbSp (and other RNAP II holoenzyme associated components) and whether the splicing defect conferred by srb.5-100 is indirect. If splicing is affected directly, this expriment could serve as a useful starting point in isolating splicing factors that are associated with the yeast RNAP II holoenzyme. For ths prcscnt. it seems likely that the srb5-100 splicing defect is indirect. One po\sthic c~plimûtionis that srb5-100 might reduce the expression of some genes that are
rcquircd for mRNA splicing. Recently, global mRNA expression patterns have been Jcicrmincd for a number of RNAP 11 holoenzyme mutants and compared to WT strains
(Holstege et al., 1998). One of the mutants assayed was a srb5-deletion strain and the rcsults of this study are consistent with the indirect hypothesis. Fifteen percent of the mRNAs in this strain were reduced by more than two-fold with respect to the WT strain. 1 searched this database of genes (Holstege et al., 1998) and found seven splicing-related genes (Table 4.2). The expression of another four genes was marginally affected and these are also inciuded in Table 4.2. Most of these genes are essential and their encoded proteins play a role before the fmt splicing step (consistent with the observed step-1 splicing defect observed in srbS-100 extracts; see Figure 4.3). It is also possible that transcription of U snRNAs is reduced, although these RNA levels were not assayed for in the Holstege et al. study (Holstege et al., 1998). None of the twelve RNAP II subunit genes was underexpressed in the Ad5 strain (Holstege et ai., 1998) consistent with the observation that RNAP II non-specific transcription is not affected in the bsrb5 or srb5-100 strain extracts. Each of the under-expressed splicing genes (Table 4.2) could be assayed by protein-blot analysis in order to determine whether the decrease in mRNA leveIs corresponds to a decrease in protein expression. The expression of mRNAs for this same set of genes (Table 4.2) was also decreased in a number of other RNAP II holoenzyme mutants, including temperature-sensitive alleles of RPBI and SRB4 (Holstege et ai., 1998). This observation suggests that extracts made from these mutants after shifung them to the non-permissive temperature might also have splicing defects (depending on whether levels of splicing factors fell in these strains during growth at the non-permissive temperature). Consistent with this idea, I found that extracts of Rpo2lp zinc-binding domain mutants also had reduced splicing activity Table 4.2: mRNA expression levels of some splicing genes are deaeased in a Asrb5 mutant with respect to a WT straîxtl gene function lst/2nd essential fold change in step (Y /N) mRNA expression in a &rbS s train2
U1 snWassociateci protein 1st in commitment complex part of Prpl9p associated 1st complex required for assembly of U4/U6.US 1st into spiiceosome associates with spliceosome 1st before 1st catalytic step U1 snRNP associated protein 1st orthologue of U1 snRNP 70 K protein U1 snRNP associated protein 1st in cornmitment complex Ul snRNP protein 1st (loosely associated) branchpoint binding protein 1st componen t of the commitment complex release of intron from spliceosome post-2nd and spliceosome disassembly potential component of tri-snRNP ? orthologue of Ul snRNP A protein 1st required for commitment complex formation
Notes to Table 4.2 1. Data in this Table from Holstege et al. 1998 are available at http:/ / www.wi.mit.edu/ young/expression-htm1 2. Numbers below mutants indicate how expression of a particular mRNA is changed with respect to a WT strain. The mutant used carrieci a deletion of the Srb5 ORF. Expression of 15% of ali genes assayed in this strain showed a greater than two-fold decreasewith respect to W. * Indicates a component of the U1 snRNP and/or a component required for commitment complex assembly. although the defect was not as great as that observed in srb5-100 extracts. Again, conflfzning that this reduction is due to a reduction in expression of splicing machuiery components would require protein-blot andysis of expression levels. In the event that splicing components were found to be at WT levels in the mutant extract, one would have to invoke alternative mechanisms to explain the decreased splicing activity observed in srb5-100 and mutant rpo2l extracts. Heat-shock treatment reduces splicing activity in both yeast and mammalian systems (Utans et al., 1992; Yost and Lindquist, 1991). This inhibition is mediated in the mammalian system by a reversible disassembly of the U4/U6.U5 tri-snRNP (Utans et al., 1992). Recovery of splicing activity in heat-shocked yeast extracts can be mediated by the addition of the heat-shock proteins HsplMp and Hsp70p (Vogel et al., 1995). It is possible that mutations in RNAP II transcription factors also cause a decrease in splicing activity through a similar mechanism. This predicts that ce11 extracts prepared from heat-shocked yeast cells or from sr65- 100 mutants might possess less assembled tri-snRNP and that reassembly might be induced by Hsp l04p and Hsp70p. The work described in this chapter also includes an attempt to show that splicing activity can be inhibited in yeast extracts by the CTD peptide. No evidence was found for this, although splicing in HeLa extracts was inhibited by the sarne peptide as has been dernonstrated (see (Ywyev et al., 1996) and Fig. 4.5). In addition, Mike Shales in Jim Ingles' group (University of Toronto) found no evidence that splicing could be inhibited in vivo by overexpressing a CTD peptide in yeast (Mike Shales; personal communication). The fact that the CTD does not inhibit splicing in yeast could be interpreted in one of three ways. First, there may be no CTD-associated splicing factors in yeast and the observed interaction between splicing factors and the mammalian CTD has not been consewed in yeast. As 1 discussed in Chapter 1, there is no evidence for CTD-associated splicing factors in yeast and it is questionable whether splicing needs to be coupled to transcription in vivo since so few yeast pre-mRNAs are spliced (less than 5% of al1 RNAP II transcribed genes). Of those yeast genes that have introns, most have only one intron that is comparatively shon (cl000 nucleotides) with well conserved splice-sites; this al1 argues against the need for factors such as SR proteins which enhance splicing efficiency and regulate alternative splicing between multiple introns.
Second, there are CïD associated spiicing factors in yeast but the CTD peptide does not disrupt their function as it does in mamrnalian systerns; the splicing-inhibition activity of
CTD peptide in the mammalian system cannot be explahed by the fact that it sirnply interacts with spiicing cornponents because it does not have to be removed fiom the extract in order to affect inhibition. Third, the CTD peptide cm inhibit splicing in yeast extract, but because the peptide is processed differently in yeast extract (folded differently or phosphorylated differently) it is not present in an inhibitory configuration. Therefore, while the meaning of these preliminary observations is questionable. it does seem to point future searches for splicingltranscription coupling factors away from using the yeast system. References
Cagas, P. M., and Corden, J. L. (1995). Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II. Proteins 2 1, 149-60.
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a sotuble extract from isolated marnmalian nuclei. Nucieic Acids Res 11, 1475-89.
Fu, X. D. (1995). The superfamily of argininekerine-rich splicing factors. RNA 1, 663-
80.
Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998). Dissecting the regulatory circuitry of a eukaryotic genome. Ce11 95,7 17-28.
Kim, E., Du, L., Bregman, D. B., and Warren, S. L. (1997). Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-rnRNA. J Ce11 Bi01 136, 19-28.
Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994). A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Ce11 77,599-608.
Koleske, A. J., Buratowski, S., Nonet, M., and Young, R. A. (1992). A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIfD. Ce11 69,883-94. Koleske, A. J., and Young, R. A. (1994). An RNA polymerase II holoenzyme responsive to activators. Nature 368,466-9.
Ling, M., Merante, F., and Robinson, B. H. (1995). A rapid and reliable DNA preparation method for screening a large number of yeast clones by polyrnerase chah reaction. Nucleic Acids Res 23,4924-5.
Moore, M. J., and Sharp, P. A. (1992). Site-specific modification of pre-mRNA: the 2'- hydroxyl groups at the splice sites. Science 256,992-7.
Studier, F. W., and Moffatt, B. A. (1986). Use of bactenophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Bi01 189, 1 13-30.
Thompson, C. M., Koleske, A. J., Chao, D. M., and Young, R. A. (1993). A muItisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Ce11 73, 1361-75.
Utans, U., Behrens, S. E., Luhrmann, R., Kole, R., and Kramer, A. (1992). A splicing factor that is inactivated during in vivo heat shock is functionally equivaient to the [U4/U6.US] triple snRNP-specific proteins. Genes Dev 6,63 1-4 1.
Vogal, J. L., Parsell, D. A., and Lindquist, S. (1995). Heat-shock proteins Hspl04 and Hsp70 reactivate mRNA splicing after heat inactivation. Curr Bi01 5,306-17.
Yost, H. J., and Lindquist, S. (199 1). Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Mol Cell Bi01 II, 1062-8. Yuryev, A., Patturajan, M., Litingtung, Y., Joshi, R. V., Gentile, C., Gebara, M., and Corden, J. L. (1996). The C-terminal dornain of the largest subunit of RNA polymerase II interacts with a novel set of serinehrginine-rich proteins. Proc Nat1 Acad Sci U S A 93, 6975-80. Chapter V Thesis summary and future directions Thesis summary and future directions
Zinc-binding domain mutants of RP021 1 have shown that seven zinc ions CO-puri@with yeast RNAP II. This number is consistent with the number of zinc-binding motifs in the twelve different subunits of the enzyme and with the ability of these subunits to bind zinc in an in vitro zinc-blotting assay
(Treich et al., 1991). One of the subunits, Rpbgp, was demonstrated to have a zinc-
stoichiometry of two, consistent with the presence of two zinc-binding motifs (Woychik et al., 1991). The methods described in Chapter 2 could be used to determine the zinc- stoichiometry of each of the remaining individual subunits that demonstrate zinc-binding ability in vitro: RpoZlp, Rpb2p, Rpb3p, RpblOp and Rpbl2p. This would serve to
independently confm that these subunits cm bind zinc and that the zinc stoichiometry of RNAP II is seven. Conservative substitutions of conserved and potentialiy zinc-coordinating arnino acids in the Rpo2lp zinc-binding domain conferred growth defects and decreased RNAP II activity in extracts (Figure 2.1, 2.5). This demonstrates that this domain is important to the function of the enzyme. It seems most likely that these mutations do not decrease the steady-state levels of Rpo2lp (Figure 2.4) and the association of each of the subunits (except for RpWp and Rpbipj with the Ml mutant RNAP II core enzyme is WT-like (Figure 2.7). Despite this, activity of the Ml core enzyme is reduced with respect to the WT indicating that mutations in the Rpo2lp-ZBD can reduce basal activity of the enzyme without dtering association of subunits required for this activity (Figure 2.7). Studies with the E. coli orthologue of Rpo2 lp (Pu) have demonstrated that the zinc- binding domain makes contact with a domain of double-stranded DNA just downstream of the active site (Nudler et al., 1996). Purified E. coli RNAP that carries a mutation in the Pu ZBD (analogous to mutant Ml in this study) is defective in RNAP II processivity and generates truncated transcripts in an in vitro transcription assay (Nudler et al., 1996). Although the RpoZlp-Ml mutant from this study decreases RNAP II activity in vivo, extracts prepared from the mutant strain are still able to accurately initiate transcription from a promoter-specific template and no truncated transcription products appear (Figure 2.5). It is possible that other factors present in the yeast mutant extract prevent the appearance of truncated products. Purified RNAP II from the yeast Ml mutant could be assayed in an in vitro reconstituted transcription assay (Sayre et al., 1992). If the Ml mutant RNAP II generated tnincated rranscripts, this assay could be used to puri@ factors fiom yeast extract that prevent the appearance of tnuicated transcription products. Such factors might include RNAP II holoenzyme components, elongation complex proteins or exonucleases.
A synthetic-lethal screen 1 used the zinc-binding domain mutant rpo21-30 (HSOY) as a starting mutation in a synthetic-lethai screen for additionai components of the RNAP LI transcription machinery. This method proved to be a successful one in that it identified an allele of SRBS (a known component of the RNAP II holoenzyme). The screen was not carried out to saturation (alleles of SRBS and GCR3 were identified only once) so it could conceivabty be used again to identiQ more components of the mRNA biogenesis pathway. Below, 1 review the benefits and pitfalls of the synthetic-lethal screen. In addition, 1 suggest a number of changes that could be made to the selection procedure in order to increase the chances of identifying more synthetic-lethal mutations that are directly related to the function of RNAP II.
Pointers to a successful synthetic-lethal screen The synthetic-lethal screening method is a powerful one; it is able to identiQ factors that rnay only play an auxiliary role in a pathway or factors that only interact weakly with other components of a pathway. This power also makes the system prone to the possibility of identiSing artefactual genetic interactions that may not be relevant to the starting mutation. For example, a screen for synthetic-lethal mutations can potentially identiw gene products that act in independent pathways; the combination of two mutations that are sufficiently debilitating by themselves may simply kill the cell. Such non-informative synthetic-lethal combinations may be avoided in several ways. First, by choosing for the synthetic-lethal screen an initia1 mutation that confers only a small growth defect under permissive conditions. Second, by choosing to work with only those synthetic-lethal alleles identified in the screen that, by themselves, confer only minor growth defects. Third, by choosing to work with those identified mutations that are allele-specific with the starting mutation. Fourth, by choosing to work with those identified mutations that are demonstrated to be defective biochemically in the relevant function or that can be clearly demonstrated to affect the same pathway.
These were principles which 1 kept in mind while designing the synthetic-lethal screen. If I were to perform this screen again I would make some alterations to the plan based on the experience that 1 gained from the first time around. A major shortcoming of the synthetic-lethal screen as I performed it, was the paucity of synthetic-lethd mutations with which 1 could choose to work and the fact that none of them were ailele-specific. 1 would remedy this by making two changes. First, 1 would simply spend more time collecting extragenic synthetic-lethal mutations.
This would increase the chances of finding mutants that had suong in vitro transcription defects. In addition, one could consider the large number of sect- putative mutations that were discarded because they grew (slowly) on 5-FOA medium. These potential synthetic- lethal mutants may have encoded informative mutations which were synthetically-slow in combination with rpo21-30. An in vitro transcription activity assay could be used to screen through such mutants to identify those that have a potential role in transcription. Second, by starting with an rpo2l mutant allele on a plasmid in an rpo2I-deletion suain, it would be possible quickly to screen through potential synthetic-lethal mutants that were synthetically Iethal with specific mutant alleles of ~021.S ynthetic-lethal mutants that were allele-specific to rpo21-30 might be more informative about the function of the RP021-ZBD. For example, one might expect to fmd mutations in the zinc-binding domain of RPBZ in such a screen since mutations in the analogous domain of A135 (second-largest subunit of RNAP 1) are able to suppress mutations in the ZBD of A190 (largest subunit of RNAP 1) in an allele-specific manner (Yano and Nomura, 1991). Additionally, such a screen might identify factors that enhance the processivity of RNAP II. If allele-specific synthetic-lethal mutations were not found, 1 would still choose to work with those synthetic-lethal mutants that (by themselves) conferred a strong in vitro transcription defect.
GCR3 and RNAP II
A mutant allele of GCR3 (gcr3-lm) was isolated from the screen as king synthetically lethal with rpotl-30 (Table 3.3). While there was reason to believe that Gcr3p rnight be associated with RNAP II, 1 found no evidence of such an interaction (Figures 3.8 and 3.9). Furthemore, the in vitro, promoter-specific transcription defect observed in gcr3-100 extracts was a small one (Figure 3.5) and 1 was unable to show that this effect is direct. The transcription defect conferred by gcr3-100 is either indirect or the CBC protein that was added to the extract was unable to compete with the endogenous gcr3-100 protein present in some non-productive complex. Since the majonty of evidence implicating Gcr3p in transcription cornes from in vitro transcription assays, it might be useful to confirm this defect (and the splicing defect; Fig. 3.10) in vivo by RNA-blot analysis of various pre- rnRNAs and their mature mRNA counterparts. Extracts of a gcr3-delete main had no observable transcription or splicing defect (Figure 3.7 and data not shown). This suggests that gcr3-100 protein interferes specifically with an script ion and/or splicing. This hypothesis would be supported by showing that Agcr3 (unlike gcr3-100) is not synthetically lethai with rpo21-30. The addition of CBC to extract made from the gcr3-delete strain would not be expected to have any effect on either transcription or splicing activity. If it is detennined that gcr3- 100 directly confers a transcription defect, genetic methods could be used to identify factors that mediate this effect. Presumably, allele-specific suppressors of gcr3-100 (that do not suppress the growth defect of Agcr3) could be isolated. These suppressors might identify factors that interact specifically with gcr3-1 ûû mutant protein and cause the observed in vitro transcription andlor splicing defects. Gcr3p has a demonstrated role in efficient splicing (Lewis et al., 1996b; Lewis et al., 1996a) and 1 hypothesized that the in vitro splicing defect observed for gcr3-100 (Figure 3.10) coupled with the transcription defect conferred by rpo2 1-30 (Figure 2.5) could be responsible for the synthetic-lethality observed between these two alleles. This hypothesis could be supported by showing that other mutant alleles of other splicing genes are also synthetically-lethal with rpo21-30. This hypothesis would also predict that Agcr3 (which does not confer an in vitro transcription defect or an in vitro splicing defect) would not be synthetically-lethal with rpo2 1-30.
GCR3
Gcr3p has a number of assayable functions that make it amenable to further study. Gcr3p interacts with the second subunit of the cap-binding complex (MudlJp) in a two- hybrid assay (Colot et al., 1996; Fields and Song, 1989). These two proteins form the cap-binding complex (CBC) which binds S-capped mRNA and is able to shift the mobility of capped mELYA in a non-denaturing gel-shift assay (Gorlich et al., 1996). Lastly, the CBC has an established role in in vitro splicing; immunodepletion of the CBC and associated factors is able to inhibit splicing activity and splicing commitment-complex formation in both yeast and mammalian extracts (Lewis et al., 1W6b; Lewis et al., 1996a). The re-addition of recombinant CBC to these depleted extracts is able panially to restore splicing activity to these extracts (Lewis et ai., 1996b; Lewis et al., 1996a).
A series of intemal deletion mutations could be made in both Gcr3p and Mudl3p to determine which domains of these proteins are responsible for the interaction of the two proteins in the CBC. The interaction between the deletion derivatives of these two subunits
could be assayed for by using the two-hybrid assay and/or by assaying the ability of His-
tagged or glutathione-S-transferase (GST) fusion derivatives to interact with one another in affinity chromatography experiments. This same set of deletion mutants could be used to Iocate the domains of the two CBC subunits that are responsible for binding capped mRNA in gel-mobility shift assays. Finally, deletion mutants of Gcr3p and Mud13p could be used
to localize those domains of the CBC that mediate splicing activity in extracts. As 1 discussed in Chapter 3, immunodepletion of CBC from extracts causes a decrease in splicing activity. This effect is probably mediated by the interaction of the CBC with other protein components of the splicing machinery (since deleùng GCR3 does not confer a splicing defect by itself). Presumably, a class of deletion mutations in the CBC exists that does not inhibit the interaction between Gcr3p and Mud l3p or between CBC and capped mRNA but does inhibit the interaction between the CBC and other protein factors that mediate efficient splicing. The entire Gcr3p or Mudl3p ORF could be used in a two- hybrid screen to identiQ splicing factors that associate with the CBC. The involvement of these factors in splicing could be confirmed by assaying splicing activity in extracts made from a strain in which the gene has been deleted. Aiternatively, CBC-interacting factors could be identified by affinity chromatography using the HisMA-tagged Gcr3p that was made in Chapter 3. This HA/His-tagged Gcr3p could be depleted from yeast extract using anti-HA monoclonal antibody. Presumably, this would deplete CBC and inhibit splicing in yeast extracts. Factors that are CO-depleted(and are responsible for mediating the CBC's stimulatory effect on splicing) could be identified by mass spectrometry. Once the general domains responsible for the various CBC functions have been identified, point mutations could be made in those domains responsible for mediating enhanced splicing in vitro. Recombinant, mutant proteins could be made in E-coli. Those mutant proteins that are able to assemble into a cap-binding complex and that are able to bind capped mRNA could be added back to CBC-immunodepleted extracts to confirm that t lwy do not cnhance spiicing activity. These mutations could then be introduced into a
J~-ri!.C;LI;I strain and assayed for growth phenotypes or for their ability to splice a reporter consirucc (Lcgrain end Rosbosh. 1989: Lesser and Guthrie, 1993). Any phenotypes confcmd by mutations could then be used to isolate second-site, allele-specific suppresors that might identify interacting factors that mediate the splicing function of yeast CBC. The involvcment of these factors could be confirmed by assaying splicing andor commitrnent- complex formation in yeast strains where the gene corresponding to the factor has ken deleted. The CBC aiso has an auxiliary role in 3kleavage of nascent pre-mRNAs in HeLa extracts ((Flaherty et al., 1997) and section 4.2 of Chapter 1 ). 3'-cleavage assays could dso be performed in yeast extract (Butler et al., 1990) made from WT, Agcr3 and gcr3-100 strains in order to determine whether the yeast CBC is also required for efficient 3'- cleavage of pre-mRNAs. Alternatively, the yeast CBC could be ïmmunodepleted hmWT extracts using the HisNA-tagged Gcr3p strain. Purified, recombinant CBC could be added to irnmunodepleted extracts in order to demonstrate that any observed effects were direct. Finally, the deletion analysis strategy discussed above could be used to identify those domains of the CBC that are responsible for any observed effects on 3'-processing. Deletion and point mutants generated in the above study could be assayed for synthetic- lethality with mutations in the RF021 gene. This may help to elucidate which CBC function is essential in the presence of a mutated Rpo2 1p. SRBS
A mutant allele of SRBS (srb5-100) was isolated fiom my screen as king synthetically lethd with rpo21-30 (Table 3.3). This factor was identified previously as a component of the RNAP iI holoenzyme (Thompson et al., 1993) and of the mediator (Koleske and Young, 1994). The identification of this factor demonstrated that the synthetic-lethal screen
was able to identiw components of the RNM II transcription machinery. However, further study of this mutant was not pursued since Srb5p had aiready been identified as a
component of the holoenzyme. Furthemore, srbS-100 did not appear to be synthetically- lethal with rpo21-30 in an dele-specific manner (Table 3.4) so it was unlikeIy that rurther study would reveal anything about the RP021-ZBD in particular. Instead, 1 chose to pursue the possibility that a mutation in srbS rnight confer an in vitro splicing defect. At the time that I made this decision, it had ken shown that the CTD peptide or antibody recognizing the CTD was able to inhibit splicing in HeLa extract (Chabot et al., 1995; Kim et ai., 1997; Vincent et al., 1996; Yuryev et al., 1996). In addition, 1 had some initial success with duplicating this effect in yeast extract (see experiments performed by Donan Anglin below). 1 considered that mediator components such as Srb5p require the CTD in order to interact with core RNAP II (Kim et al., 1994; Thompson et ai., 1993) and that the absence of this factor might dismpt splicing activity in vitro in a way related to CTD peptide disruption of splicing in vitro. Extracts made from sr65100 were indeed deficient in splicing activity (Figure 4.3). However, 1 was unable to restore splicing activity by adding recombinant SrbSp and Srb2p to the mutant extract, suggesting that the spiicing defect was an indirect one (Figure 4.4). The indirect hypothesis could be supported by showing that irnmunoprecipitation of HA-tagged SrbSp from WT extracts does not reduce splicing activity. A Asrb5 mutation is associated with a two-fold decrease in the expression of approximately 15% of yeast mRNAs (Holstege et al., 1998). Included in these are the mRNAs that encode a number of splicing components (see Table 4.2). These decreases could explain the splicing defect observed in srb5-100 extracts. This hypothesis could be strengthened by showing that the decreased mRNA expression in this strain corresponds to a decrease in the gene product using protein-blot experiments. This hypothesis also predicts that mutations in other transcription-related genes might confer in vitro splicing defects. 1 found that extracts made from the RP021-ZBD mutants were also deficient in splicing activity (although to a lesser degree than srb5-100 extracts) (Figure 4.7). Presumably, such an indirect effect would not be specific to ZBD or to RP021 mutants. Extracts could be made from other RfO2l mutants or fiom other SRB mutants and assayed for splicing activity in order to confithis.
The RNAP II CTD and splicing Recent work by other groups has demonstrated that overexpression of the CTD in HeLa cells (Kim et al., 1997) or addition of CTD peptide to HeLa extracts (Yuryev et al.,
1996) is able to inhibit splicing. 1 wanted to determine whether this sarne effect could be repeated in the yeast system. A mouse CTD fusion protein consisting of 52 CTD repeats
(a gift from Jim Ingles and Raj Gupta) was fust assayed for inhibitory activity by Donan Anglin (data not shown). Mouse CTD protein was coupled to Affi-Gel beads which were
incubated with yeast extract and then pelleted before taking an aiiquot of the supernatant to assay for splicing activity. CTD coupled to beads was found to inhibit yeast splicing activity, while beads alone had no affect (data not shown from Dorian Anglin). Furthemore, in a separate experiment, beads coupled to were able to deplete extract of splicing activity and a 1 M NaCl eluate from the beads was able to restore splicing activity while eluate from a beads-alone column had no restorative activity (data not shown). These preliminary results partly formed the bais for assaying splicing activity in srb5-100 extracts (see above). Unfortunately, 1 was unable to repeat these results in subsequent triais using other yeast extracts and batches of CTD-beads. For this reason, 1 decided to repeat the CTD-peptide inhibition experiment that was fmt reported by Corden's group (Yuryev et al., 1996)- A synthetic 8-mer CïD peptide was synthesized and added to
HeLa extract (as a positive control for inbibitory activity) and then to yeast extract (to ask if this same peptide could inhibit yeast spiicing). While 1 was able to show that the CTD peptide inhibited splicing activity in HeLa extract (Figure 4.3, the same peptide had no effect on yeast extract splicing activity (Figure 4.6). The CïD peptide may not inhibit yeast splicing for a number of reasons. Yeast splicing factors may not be coupled to the yeast CTD at al1 or in a way that maices splicing activity resistant to CTD peptide. Altematively, factors present in the yeast extract (or not included or not active in the extract as it was prepared) might prevent the CTD from inhibiting splicing activity. Given the number of possibilities, it is probably more worthwhile to approach the problem in some other way. Three types of experiments could help determine whether splicing factors are coupled to RNAP II via the CTD. Fust, affinity chromatography has been used to purify human proteins that interact with the full-length mouse RNAP II CTD (Emili, 1997). Two proteins identifïed by this method include the essential human splicing factor PSF and p54nrb (Emili, 1997; Gozani et al., 1994; Straub et al., 1998). The GST-CTD fusion protein described in Chapter 4 (Patturajan et al., 1998) could be used in a similar manner to isolate proteins from yeast extract that specifically interact with the CID. Proteins that are increasingly depleted fiom extract by increasing concentrations of GST-CTD ligand but not by GST alone could be identified by denaturing polyacrylarnide gel electrophoresis and silver staining. Individual bands that bind to the GST-CTD colurnn could be excised from the stained gel, and prepared for mass spectrometry . Second, RNA polymerases 1 and iII lack a sequence that is similar to the RNAP II CTD. If splicing is coupled to transcription via the RNAP II CTD, one would predict that intron-containing genes wodd not be efficiently spiiced if they were transcribed by RNA polymerases I or m. This has been shown to be the case in mamrnalian cells. Intron- containing genes are not spliced when transcribed by RNA polymerase III in human cells (Sisodia et ai., 1987; 'White and Kunkel, 1993). A mRNA-intron can be spliced from an
KVAP III tRNA transcript in yeast aithough the efficiency of this splicing event was not addressed (Kohrer et ai., 1990). This type of system could be used to determine whether the CI?) is able to couple splicing to transcription. First, splicing of an intron-containing gene transcribed by RNAP 1 or IiI could be monitored by RNA-blot analysis of RNA extracted from a reporter yeast strain. Second, a chimaenc fonn of RNAP 1 or III couid be constructed in which the RNAP II CïD was fused to the C-terminus of the largest subunit. This chimaeric-largest subunit (or a WT control) could then be expressed in the yeast reporter system (possibly under an inducible promoter if the chirnaeric constnict proves to be toxic to the cell). One would expect that the ratio of spliced to pre-mRNA would increase if the CTD were able to recmit splicing factors to the transcribing poiymerase. Third, random point mutations could be constructed in the yeast RNAP II CTD and then subcloned to the RP021 ORF and introduced into a WT strain or into an RP021- deletion strain carrying RP021 on a URA3 maintenance plasmid. These mutants could be screened for those that have linle effect on in vitro transcriptional activity (as detected by viability of the mutant strain and expression of a P-galactosidase reporter gene) but that have reduced splicing activity (as detected by expression of a CUPl reporter gene containing an intron; proper expression of CUPI confers resistance to higher levels of copper in the growth medium (Lesser and Guthrie, 1993)). The detection of such mutants would support (but not prove) the idea that splicing factors associate with the RNAP II CTD. Associated splicing or coupling factors could then be identified by selecting for allele-specific suppressors of the CïD mutants identified in the fmt step. References
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