Order Number 8913650
In vitro studies of self-splicing group II introns
Hebbar, Sharda Kattingeri, Ph.D. The Ohio State University, 1989
Copyright ©1989 by Hebbar, Sharda Kattingeri. All rights reserved.
U-M-I 300N.ZecbRd. Ann Arbor, MI 48106
IN VITRO STUDIES OF SELF-SPLICING GROUP II INTRONS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Sharda Kattingeri Hebbar, B.S., M.S.
*****
The Ohio State University
1989
Dissertation Committee: Approved by
P. A. Fuerst
L. F. Johnson — A. M. Lambowitz * Adviser P. S. Perlman Department of Molecular Genetics Copyright by Sharda Kattingeri Hebbar 1989 To My Parents, and To My Husband, Raja. ACKNOWLEDGEMENTS
I would like to thank Dr. P.S. Perlman for his assistance and guidance. Special thanks go to Kevin Jarrell and Rosemary Dietrich not only for their technical advice but also for their friendship. To my husband, Raja, I would like to express my sincere gratitude for his encouragement, support and endurance throughout my endeavors.
iii VITA
February 21, 1960 ...... Born - Andhra Pradesh, India
1980 ...... B«S., Osnania University, Hyderabad, India
1980 - 1982 ...... M.S., Andhra University, Waltair, Andhra Pradesh
1983 - 1986 ...... Graduate Teaching Associate, MCDB Program, The Ohio State University, Columbus, Ohio
1986 - 1988 ...... Graduate Research Associate, Department of Molecular Genetics, The Ohio Sta+e University, Columbus, Ohio
FIELDS OF STUDY
Major Field: Molecular Genetics TABLE OF CONTENTS
INTRODUCTION ...... 1
I.A. Catalytic RNA ...... 1 I.B. Yeast mitochondrial DNA ...... 2 I.B.l. Mosaic genes 2 I.B.2. Maturases 3 I.B.3. Group I introns 5 I.B.3.a. Discovery of RNA catalysis 6 I.B.3.b. Tetrahymena rRNA intron self-splices by a two step transesterification pathway 7 I.B.3.C. Tetrahymena rRNA intron is a group I intron 9 I.B.3.d. Mitochondrial group I introns self-splice in vitro 1 0 I.B.3.e. Not all group I introns are self splicing 11 I.B.3.f. The intervening sequence RNA of Tetrahymena is an Enzyme 12 I.B.3.g. Active site model 13 I.B.4. Group II intron splicing ...... 14 I.B.4.a. Conserved sequence elements and secondary structure ...... 14 I.B.4.b. Group II introns in yeast mitochondria ...... 16 I.B.4.C. Group II introns encode proteins related to reverse transcriptase...... ,, 17 I.B.4.d. AI5g self-splices in vitro by a n o v e l ...... 18 I.B.4.e. Role of branch formation ...... 19 I.B.4.f. Group II intron splicing resembles nuclear pre-mRNA splicing ...... 20 I.B.4.g. Alternative reaction conditions ...... 2 2 I.B.4.h. Trans-splicing ...... 23 I.B.4.i. Dependance on 5’ exon sequences...... 25 I.B.4.j. Multiple 5’exon binding sites ...... 26 I.B.4.k. Domain 4 is dispensible ...... 26 I.B.4.1. Domain 5 is required for 5’ exon release...... 27 I.C. Dissertation Goals ...... 28 v MATERIALS AND METHODS 29
II.A. Plasmid constructions ...... 29 II.B. Plasmid preparation...... 30 II.C. Transcription and Purification of R N A ...... 30 II.D. All Splicing reactions...... 30 II.E. Site-directed mutagenesis...... 31 II.F. Purification and end-labeling of oligonucleotides ...... 31 II.G. RNA sequencing...... ,....32 II.H. Northern B l o t s ...... 32 II.I. 3* end-labeling of R N A ...... 32 II.J. Limit T1 d i g e s t ...... 32 U.K. Other Methods...... 33 II. L. Enzyme reagents...... 33
RESULTS...... 34
III.A. Test of the generality of group II self splicing ...... 34 III.A.I. Cloning of intron 1 of the COX I gene ...... 35 III.B. Features of the all self-splicing reaction ...... *...... 36 III.B.1. All RNA is inactive in the standard aI5g splicing buffer ...... 36 III.B.2. All has an absolute requirement for monovalent cations...... »...... 37 III.B.3. All RNA has a higher threshold for magnesium than aI5g ...... 38 III.B.4. NH4C1 as the standard splicing b u f f e r ...... 39 III.B.5. All reactions have a temperature optimum of 4QoC ...... 40 III.B.6 . The reaction is unimolecular and time dependent ...... 40 III.B.7. Optimum reaction conditions ...... 41 III.C. Characterization of the NH4C1 reaction products 42 III.C.l. Identification of products containing the 3 * ex o n ...... 43 III.C.2. First test of the validity of the assignments...... 44 IiI.C.3. Accurate ligation of exons ...... 45 III.C.4. The slowly migrating RNA species is excised intron lariat (IVS-LAR) ...... 46 III.C.5. Accurate 5 ’ Cleavage ...... 47 III.C.6 . Technical problems in mapping the branch point ...... 48 III.C.7. Summary and implications of product characterization ...... 48 vi III.D. All yields some novel reaction products in KC1 49 III.D.1. Further analysis of KC1 effects on the all reaction ...... 50 III.D.2. Characterization of novel KC1 products ...... 51 III.D.3 A proposed pathway for the KC1 reaction...... 54 III.E. Spliced exon reopening (SER) 55 III.E.l. All does not reopen spliced exons ...... 55 III.E.2. SER is sequence specific...... 57 III.F. Much of the intron ORF can be deleted 58 III.F.l. Location of the branch site in the shortened IVS-LAR...... -...... 60 I1I.G. Studies using portions of all 62 III. G. 1. Trans-splicing ...... 62 III.G.2. The conserved 5*boundary sequence is needed for trans-splicing...... 64 III.H. Summary 6 6 III.I. Studies of aI5g self-splicing - 67 111.1.1. Introduction...... 67 111.1.2. Domain 5 is required, in cis, for the second step of splicing...... 6 8 111.1.3. Mutational analysis of the conserved 5* end of the intron ...... 71 111.1.3.a. The first 7 nt of the intron are essential for branch formation ...... 71 111.1.3.b. 5’ end of the intron plays a role in S E R ...... 75 111.1.4. Domain 6 is not required for splicing in v i t r o ...... 76 III.J. Molecular dissection of domain 5 77 III.J.l. Introduction...... 77 III.J.2. Heterologous experiments...... 77 III.J.3. Mutations of the highly conserved unpaired regions of domain 5 ...... 78 III.J.3.a. The 4 base pair loop ...... 78 III.J.3.b. RNA truncated at the Rsa I site in domain 5 is inactive...... 80 III.J.3.0. The CG bulge ...... 81 III.J.4. Bottom helix ...... 83 III.J.5. 7 nt in the 5* half of domain 5 can form a perfect match with 7nt in domain 1 ...... 85
DISCUSSION 8 8
IV.A. All self-splices in vitro 8 8 IV.A.l. All self-splices under conditions different from those of aI5g and bll ...... 90 IV.A.2. All reaction pathways ...... 91 IV.A.3. All undergoes a post-splicing vii reaction in K C 1 ...... IV.A.4. All does not carry out apliced exon reopening ...... IV.A.5. Much of the intron open reading frame can be deleted...... 93 IV.B. 5* end of the intron is not required for 5' exon release but plays a role in branch formation 94 IV.C.. 5* end of the intron plays a role in SER 96 IV.D. Lariat formation is not required for efficient splicing in vitro 96 IV.E. Domain 5 is also required for the second step in splicing 97 IV.E.l. 4 base GAAA loop is not critical for domain 5 function ...... IV.E.2. The 2 base bulge is crucial for domain 5 function ...... IV.F. Future experiments 100 IV.F.l. Point of interaction...... IV. F. 2. Transformation experiments ......
viii LIST OF FIGURES
Figure 1. Conserved Sequences in Nuclear, Group I and Group II introns ...... 102 Figure 2. Mosaic Genes in Yeast mtDNA...... 104 Figure 3. Transesterification Mechanism for the Tetrahymena IVS...... 106 Figure 4. The Internal Guide Sequence (IGS)...... 108 Figure 5. The Single "Active Site" or the "Guide Sequence" Model...... 110 Figure 6 . Secondary Structure Model of a Group II intron (aI5g)...... 112 Figure 7. Splicing Mechanism of Nuclear mRNA Precursors...... 114 Figure 8 . Comparison of Group I, Group II and Nuclear Intron Splicing...... 116 Figure 9. Group II Intron Splicing Pathway...... 118 Figure 10. Plasmid Construction...... 120 Figure 11. In Vitro Reactivity of the all Precursor RNA...... 122 Figure 12. Effect of Different Salts on the In Vitro reaction...... 124 Figure 13. Concentration of Magnesium Chloride...... 126 Figure 14. Concentration of NH«C1...... 128 Figure 15. Optimum Reaction Temperature...... 130 Figure 16. Time Course of the NH«C1 Reaction...... 132 Figure 17. Fate of the downstream exon...... 134 Figure 18. Analysis of 3' Ends of Precursor and Products...... 136 Figure 19. Northern Blot Analysis...... 138 Figure 20. Primer Extension on 5'E-3'E and 5*E RNAs...... 140 Figure 21. Sequencing of Spliced Exons...... 142 Figure 22. 3* End-Labeling of the NH«C1 reaction product...... 144 Figure 23. Debranching Experiment on the NH4 CI IVS-LAR...... 146 Figure 24. Mapping the 5’ Ends of the IVS-LAR and IVS-BL...... 148
ix I M i
Figure 25. Concentration of K C 1 ...... 150 Figure 26. Time Course of the KC1 Reaction...... 152 Figure 27. Mapping the 5’ ends of the Novel Linear RNAs in KC1...... 154 Figure 28. Gel Purification and Re- electrophoresis of the Novel Non- Linear RNA Species in KC1...... 156 Figure 29. 3* End-Labeling of Non-Linear RNAs in KC1...... 158 Figure 30. Reaction pathway in EC1 ...... 160 Figure 31. Proposed Model for the Post-Splicing Reaction in KC1...... 162 Figure 32. Evidence for the Post-Splicing Reaction...... 164 Figure 33. All Does Not Carry Out Spliced exon reopening...... 166 Figure 34. Heterologous SER experiment...... 168 Figure 35. In Vitro Reactivity of the Shortened all Intron...... 170 Figure 36. Treatment of the Shortened IVS-LAR RNA with the Debranching Extract...... 172 Figure 37. Limit T1 Digest on the IVS-LAR and the IVS-BL RNA...... 174 Figure 38. Schematic Representation of the "Half Molecules" used in the Trans-Splicing Reactions...... 176 Figure 39. All Frecursor RNA Interrupted Within Domain 4 Trans-Splices in vitro...... 178 Figure 40. Most of Domain 4 is not Required for In Vitro Trans-Reactions...... 180 Figure 41. The Conserved Boundary Sequence is Required for Trans-splicing...... 182 Figure 42. Sequence Analysis of Domain 5 Deletion...... 184 Figure 43. Domain 5 is Required for the Second Step in Splicing...... 186 Figure 44. Domain 3 is required for 5* cleavage...... 188 Figure 45. Proposed Model for Domain 5 Interaction...... 190 Figure 46. The FIB IVS-LARs are not Susceptible to the Debranching Extract...... 192 Figure 47. Sequence Analysis of 5' intron Pst I Mutation...... 194 Figure 48. In Vitro Analysis of pSH5’I-Pst I RNA...... 196 Figure 49. pSH5’I-Pst I RNA is blocked in SER...... 198 Figure 50. Sequence Analysis of Domain 6
x Deletion...... 200 Figure 51. Domain 6 is not Required for Splicing In Vitro...... 202 Figure 52. Structure of Domain 5 ...... 204 Figure 53. Comparison of all and aI5g domain 5 ...... 206 Figure 54. Domain 5 of Al5g Cleaves At or Near the 5' Splice Site of All...... 208 Figure 55. Sequence Analysis of the Bam HI and the Bam-4 Mutation in Domain 5 ...... 210 Figure 56. In Vitro Analysis of the Bam HI and the Bam-4 RNA...... 212 Figure 57. Schematic representation of the run off experiment 214 Figure 58. RNA Truncated at Rsa I Does Not Release free 5 ’exon ...... 216 Figure 59. Sequence Analysis of the 2 base Bulge Mutants...... 218 Figure 60. In Vitro Analysis of the Bulge Mutants...... 220 Figure 61. Schematic Representation of Mutations in the Bottom Helix of Domain 5...... 222 Figure 62. Sequence Analysis of the Bottom Helix mutants...... 224 Figure 63. In Vitro Analysis of the Bottom Helix mutants...... 226 Figure 64. Domain 5, in Trans, Partially Supresses the Mutant Phenotype of the Bottom Helix Mutants...... 228 Figure 65. Working Model for Domain 5 Interaction...... 230 Figure 66. Point Mutants in Domain 5 ...... 232 Figure 67. Sequence Analysis of the point mutants in domain 5...... 234 Figure 68. In Vitro Analysis of the Point Mutants...... 236 Figure 69. Sequence Analysis of the Domain 1 Mutation...... 238 Figure 70. In Vitro Analysis of the Domain 1 mutant RNA...... 240
xi CHAPTER I
INTRODUCTION
I.A. Catalytic RNA
Since their discovery in 1977, "introns" (Berget et al., 1977) have been found in many eukaryotic and in occasional prokaryotic genes. Based . on their nucleotide sequence, introns fall into four major groups: nuclear pre- mRNA introns, nuclear tRNA introns, group I and group II introns (summarized in Figure 1). Whatever their origin or possible function, all introns have to be removed precisely from primary transcripts by a cleavage-ligation reaction termed "RNA splicing". Splicing is a process that demands remarkable accuracy, even a small error would be intolerable, resulting in an RNA that encodes a non functional protein. How do introns achieve this high degree of specificity? Recent findings suggest that there are several mechanisms for intron excision, ranging from
"simple" RNA catalysis to an extensive reliance on proteins
(Sharp, 1987). The existence of RNA catalysis was demonstrated first and unequivocally by the discovery that accurate splicing of the rRNA precursor of Tetrahymena ihermophila occurs in the absence of proteins (Cech et al., 1 2 1981; Kruger et a L , 1982). The ability of the Tetrahymena intron to "self-splice" implies that both the structural and catalytic activities required for splicing are inherent in the structure of the RNA. This startling finding that
RNA molecules can act as catalysts upset the long-standing dogma that all enzymes are proteins. Though RNA catalysis has only recently come to light, three major classes of catalytic RNAs can be delineated on the basis of reaction mechanism: self-splicing group I and group II introns,
RNase P RNA and self-cleaving RNAs. The major emphasis of this dissertation will be on self-splicing group II introns in yeast mitochondria.
I.B. Yeast mitochondrial DNA
I.B .1. Mosaic genes
Bakers yeast, Saccharomyces cerevisiae, is a facultative aerobic organism that can tolerate mutations in
its mitochondrial genome, when grown on fermentable carbon sources. This feature has been exploited to introduce mutations into the mitochondrial DNA to identify mitochondrial genes and to study gene expression. The mitochondrial genome (8 80 kilobase pairs in length) encodes a limited set of proteins and RNAs, essential for mitochondrial protein synthesis, several subunits of inner membrane enzyme complexes required for functioning in the respirarory chain and a number of proteins involved in mRNA processing (reviewed by Dujon, 1981). 3 In yeast mt DNA there are thirteen introns (Figure 2), one in the 21 S rRNA gene (Dujon, 1980), five in the cob gene encoding the apoprotein of cytochrome b (Nobregga et al., 1980; Lazowska et al., 1980) and seven in the oxi3 gene encoding subunit I of the cytochrome c oxidase (Bonitz et a l ., 1980; Hensgens et al., 1983). Many of the introns are optional and their absence in some S. cerevisiae strains is without any detectable phenotypic effect
(Dhawale et al. , 1981; Hensgens et al., 1983). Yeast mitochondria provide a very powerful model system for studying the role of intron sequences in RNA splicing events because their introns can be subjected to detailed mutational and biochemical analyses.
I.B.2. Maturases
Genetic analysis of the cob gene revealed several mutations, located in the introns, that are trans-recessive in complementation tests. This observation suggested that some mitochondrial introns specify trans-acting factors
(Lamouroux et al., 1980). Sequence analysis established that ten out of thirteen introns contain long open reading frames (ORFs), in phase with the preceding exon (Nobregga et al., 1980; Bonitz et al., 1980). In a now classical paper, Lazowska et a l ., (1980) proposed a model for the involvement of these intron-encoded proteins (RNA
"maturases") in splicing. They presented strong genetic evidence that the protein maturase encoded by intron 2 of 4 the cob gene is required for excision of the same intron.
Once made the "maturase" catalyzes the excision of the intron sequence that encodes it, thus destroying the RNA sequence that directed its synthesis. Subsequently, it was demonstrated that in addition to cob intron 2, intron 4 of the cob gene (Anziano et al., 1982; De la Salle et al.,
1982; Weiss-Brummer et al., 1982) and introns 1 and 4 of the COX I gene (Dujardin et al., 1982; Carignani et al.,
1983) are also endowed with maturase activity.
Recently strong support for the existence of the maturase protein has come from the demonstration that mit- mutants (respiratory deficient point mutants) with a mutation in the bI4 reading frame can be complemented by transformation with a plasmid encoding a fusion protein, consisting of a mitochondrial import signal linked to sequences encoding the bl4 maturase (Banroques et al.,
1986). Transformants were respiratory competent, indicating that the fusion protein is able to compensate for the deficiency in maturase activity. All these experiments show that RNA maturases are essential for RNA splicing but do not explain the mechanism of their activity. Because the intron encoded "maturases are for the most part intron-specific, it is unlikely that they are general splicing enzymes (Lazowska et a l ., 1980). The ability of some precursor RNAs to self-splice (see below) makes a direct catalytic role for maturases less likely and 5 suggests a function in stabilizing the secondary and tertiary structure of RNA.
Analysis of several cis-dominant splicing defective mutations led to the discovery that mitochondrial introns contain several short sequence elements whose integrity is required for accurate splicing (De la Salle et al., 1982;
Anziano et al., 1982; Lamb et al., 1983; Netter et al.,
1982). One particularly striking fact to emerge from this work on mutants is that, in contrast with nuclear introns, sequences within mitochondrial introns are important for
RNA splicing. Complementary base-pairing between some of these conserved sequence blocks was used to construct secondary models of intron RNA (Davies et al., 1982; Michel et al., 1982). Based on these findings, mitochondrial introns were classified as belonging to either group I or
II.
I.B.3. Group I introns
Group I introns include the majority of funga! mt DNA introns, the nuclear rRNA introns of Tetrahymena and
Physarum, certain chloroplast introns and introns in bacteriophage T« (Cech et al., 1986). Features typical of group I introns are summarized in Figure 1: 1) The 5* and
3* splice-site of group I introns are always preceded by U and G, respectively, 2) Group I introns contain short conserved elements termed box9L, box2, A and B. Another pair of conserved elements box 9R and 9R’ are always 6 present. Although the primary sequence of these two elements varies, they are always complementary. The six sequence elements almost always occur in the same polarity along the intervening sequence (IVS), 5 ’ -9R’-A-B-9L-9R-2-
3 *. Based on phylogenetic sequence comparison and computer modeling it was shown that these elements interact via pairing of complementary bases: box9L.box2, A.B and
9R'.box9R to form a conserved secondary core structure required for splicing (Michel et al., 1982; Davies et al.,
1982). In the case of cytochrome b intron 4 there is strong genetic evidence for the box9L. box2 and box 9R.9R’ interactions (Wiess-Brummer et al., 1983; Holl et al.,
1985). 3) All group I introns have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5’ and 3’ splice sites to align them precisely for splicing (Davies et al., 1982). Proof that the 5* exon.IGS interaction actually exists was provided first by genetic means (Perea et al., 1985).
I.B.3.a. Discovery of RNA catalysis
The initial discovery of catalytic RNA was made by Tom
Cech and his colleagues while studying the transcription and processing of the 26S ribosomal RNA in the protozoan
Tetrahymena thermophila. Every copy of the nuclear 26S rRNA gene is interrupted by a 414 nucleotide intervening sequence (IVS) which must be excised in order to generate the mature RNA molecule (Din et al., 1979 ). The IVS is 7 excised as a discrete linear molecule, which is subsequently converted to a circular form (Grabowski, et al., 1981). An attempt to identify the enzymes responsible for the processing led to the startling realization that the reaction is autocatalytic — purified 26S precursor RNA underwent precise intron excision in vii^o, in the complete absence of protein, requiring only guanosine, Mg*♦ and NH«+
ions (Cech et al., 1981). Rigorous proof that the intron self-excision is an intrinsic property of the intron and is not due to some tightly associated protein with splicing activity was provided by demonstrating that pre-mRNA transcribed in vitro from a recombinant plasmid undergoes the same autoexcision reaction (Kruger et al., 1982).
I.B.3.b. Tetrahymena rRNA intron self-splices by a two step transesterification pathway
An important clue to the nature of the self-splicing reaction was that a guanosine, not encoded by the DNA sequence of the gene, was added to the 5* terminus of the
IVS during its excision from the precursor RNA (Zaug et al. , 1982). This explains the requirement for guanosine
(GMP, GDP and GTP are equally effective) in the in vitro reaction. This requirement is specific and none of the other nucleosides or nucleotides containing A, C or U promote the reaction. No external energy as is provided by
ATP or GTP hydrolysis is needed for exon ligation following excision of the IVS (Kruger et al., 1982). This is a explained by a two step transesterification reaction (Zaug et al., 1982; 1983). In the first, the 3*-hydroxyl of the free guanosine acts as the nucleophile, attacking the phosphate at the 5* splice site, transferring the G to the
5* end of the IVS and leaving a 3*-hydroxyl group at the end of the 5’ exon {Figure 3). The 3*-hydroxyl group of the free 5*exon then acts as the nucleophile in the second transesterification reaction, resulting in exon ligation with concomitant release of the IVS. In the course of the reaction there is no net change in the covalent bond energy since two phosphodiester bonds are made (the G addition and exon ligation), and two are broken (the 5* and 3* splice sites). RNA cleavage and ligation activity are, therefore, intrinsic to the IVS RNA.
After its excision, the linear IVS undergoes a series of cyclization and site-specific hydrolysis reactions (Zaug et al., 1984). The final product of these intramolecular reactions, L-19 IVS RNA, is a stable, linear molecule lacking the first 19 nucleotides of the excised intron.
The lack of further reactivity of the L-19 IVS RNA is an indication that all potential reaction (or cyclization) sites on the molecule that could reach its active site have been consumed and the RNA cannot undergo additional intramolecular reactions. 9
I.B.3.C. Tetrahymena rRNA intron is a group I intron
Soon after the discovery of the self-splicing mechanism, the first hint of the generality of RNA self splicing came with the finding that the Tetrahymena rRNA intron bears a striking resemblance to sequences within group I introns (Cech, et al., 1983). The six sequence elements described above occur in the Tetrahymena intron in the same 5'- 3* polarity as in the group I introns. For the Tetrahymena IVS, there is strong evidence that pairing of elements 9L and 2 is required for self-splicing (Burke et a l ., 1986 ).
The Tetrahymena IVS, like group I introns, also contains an internal guide sequence (IGS), located at the
5* end of the intron, that acts as an adaptor molecule to align the 5' and 3* exons for splicing (Figure 4).
Subsequent deletion analysis and site-directed mutagenisis experiments with Tetrahymena rRNA intron demonstrated that this interaction is essential for self-splicing and accurate 5* splice-sifce selection (Been et al., 1986;
Waring et al., 1986). The region of the IGS proposed to interact with the 3* exon, however, appeared dispensible for splicing in vitro (Been et al., 1985). 10
I.B.3.d. Mitochondrial group I introna self-splice in vitro
Since the Tetrahymena rRNA intron is a group I intron, it seemed likely that all group 1 introns may have a similar splicing mechanism. The first evidence that mitochondrial group I intron splicing occured by the same mechanism as Tetrahymena rRNA was provided by Garriga et al., (1983, 1984). Since then, several mitochondrial group
I introns in yeast and Neurospora and introns in bacteriophage T4 have been shown to self-splice in vitro
(Garriga et a l ., 1984; Tabak et al., 1984; Van der Horst et al., 1985; Tabak et al., 1987; Partono et al., 1988; Chu et al., 1986; Gott et a l ., 1986).
In the case of the yeast mitochondrial group I introns a rather puzzling situation is encountered. Apart from the expected products, a number of aberrant products are found including lariat RNAs (Arnberg et al., 1986). Not only do they arise during the splicing of large rRNA precursor but have also been observed among the splicing products of aI3 and aI5 (Tabak, et al., 1987). Other aberrant products include a 3’ exon with an extra G covalently attached to its 5* end (resulting from attack at the 3’ splice-site) and interlocked circles of sub-intronic fragments (Tabak et al., 1987). 11
I.B.3.e. Not all group I introns are self-splicing
Although several of the group I introns are self splicing there is strong genetic and biochemical evidence for the involvement of both mitochondrial and nuclear proteins in mitochondrial RNA splicing (Lazowska et al.,
1980; Anziano et al., 1982; Faye et al., 1983; Hill et al.,
1985; Krieke et al., 1986; Schmelzer et al., 1986;
Labouesse et al., 1985; Akins et al., 1986; Herbert et al.,
1988). It is likely that in the absence of proteins some group I introns cannot fold into the active conformation necessary for self-splicing. This appears to be the case for the Neurospora large rRNA intron, which splices by G- addition in vivo (Garriga et al., 1983) but does not self splice in vitro.
Neurospora cob intron 1 self-splices in vitro but splicing of this intron is defective in the nuclear mutant cytl8 -l (Collins et al., 1985). This clearly indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo. Akins and Lambowitz (1986) recently reported that the protein encoded by the cytl8 gene is mitochondrial tyrosyl-tRNA synthetase. Additional support is provided by
Slonimski and coworkers who show that the yeast NAM2 protein, a mitchondrial leucyl-tRNA synthetase, is involved in mitochondrial mRNA splicing (Herbert et al., 1988). 12
I.B.3.f. The intervening sequence RNA of Tetrahymena is an
Enzyme
Three years after the discovery of self-splicing Tom
Cecil* s group reported another surprising finding, the stable L-19 1VS RNA was capable of acting as a true enzyme, catalyzing cleavage-ligation (intermolecvilar) reactions on exogenous RNA substrates. When provided with oligo
(cytidylic acid) as a substrate, the L-19 IVS RNA acts as an enzyme with nucleotidyltransferase (poly(C) polymerase) and phoshphodiesterase (ribonuclease) activities (Zaug et al., 1986a). With 3 *-phosphorylated oligo(C) substrates, the same ribozyme acts as a phosphotransferase and an acid phoshatase (Zaug et al., 1986b). In addition to these activities, the L-19 IVS RNA can also act as an endoribonuclease, catalysing the cleavage of large RNA molecules at sequences resembling the 5’ splice site (Zaug et al., 1986). The sequence specificity approaches that of
DNA restriction endonucleases. Site specific mutations of the enzyme active site alters the substrate specificity in a predictable manner, so that endoribonucleases can be synthesized to cleave at a variety of tetranucleotide sequences. 13
I.B.3.g. Active site model
Trans-splicing experiments involving certain oligonucleotides with sequences resembling those at the 3* end of the 5* exon (Inoue et al., 1985; Garriga et al.,
1986), suggested the existence of a 5* exon binding site in agreement with IGS model (Waring et al., 1983). . This internal binding site was proposed to be involved in specifying the 5* splice-site and, following the first step of splicing, in holding the 5* exon in place for exon ligation (Garriga et al., 1986).
The idea of a single active site mediating the self processing reactions of the Tetrahymena intron is shown in
Figure 5. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence
(5* exon), a G residue within the 5* exon binding site
(IGS) and another G residue (either a free guanosine residue or the 3’ terminal G of the IVS) that acts as the attacking group in the transesterification reactions. A conformational change (translocation) is required to bring different oligopyrimidine sequences into the active site for attack by guanosine (Cech et al., 1986).
Consistent with the active site model single base changes in either the 5* exon sequence or the 5* exon binding site altered the specificity of the self-splicing reaction (Waring et al., 1985; Been et al., 1986).
Sequence alterations in the 5’ exon binding site also 15
Intron domain 5 is highly conserved and consists of a 34 nt
sequence, that can be invariably folded into a 14 basepair hairpin, with an almost constant GAAA terminal loop and a
CG bulge on its 3* side (Michel et a l . , 1982). While
domain 6 , also known as the branch helix, is conserved in
position, domain 4 is quite variable in length (Michel et
al., 1983). Among group II introns that contain an ORF, most of it is present within domain 4 (Keller et al.,
1985).
Since the neighbouring exon sequences were not
included in the original group II intron secondary
structure models (Michel et al., 1982; Davies et al.,
1982), it had become necessary in the light of recent
trans-splicing experiments (see below), to reconsider the
secondary and tertiary interactions between group II
introns and their exons. A systematic search for conserved base-pair interactions (Jacquier et al., 1986; Michel et
al., 1987) between sequences surrounding the splice-
junctions and the single-strand terminal or internal loops
in the secondary structure models identified four potential
pairings (Figure 6 ).
First, all group II introns examined show an EBS1-IBS1 pairing. In this pairing the last five to eight
nucleotides of the 5’ exon (IBS1, intron binding site 1) are always complementary to a stretch of six nucleotides within the intron (EBSl, exon binding site 1). The second 14 changed the specificity of two other reactions: intermolecular exon ligation (trans-splicing) and the enzymatic nucleotidyl transferase activity. Thus the 5* exon binding site acts as part of the active site for pre- rRNA self-splicing, trans-splicing and the L-19 IVS RNA enzyme activity.
Having introduced self-splicing group I introns, I will now turn to splicing in group II introns which is the major topic of this dissertation.
I.B.4. Group II intron splicing
I.B.4.a. Conserved sequence elements and secondary structure
Group II introns are found in mitochondrial genes of fungi and higher plants and in some chloroplast genes, but their occurance is less wide spread than group I introns.
Group II introns lack the characteristic group I intron consensus sequences but like nuclear introns they are characterized by consensus sequences at their boundaries,
5* GUGYG ... AY 3 ’ (Michel et al., 1983; Keller et al.,
1985). In addition, all group II introns have a highly conserved 34 nucleotide sequence near their 3* ends.
As shown in Figure 6 , all group II introns can be folded into a "core" secondary structure of 6 helical domains (numbered 1-6, 5'-3*) radiating from a central wheel, which brings the 5* and 3* intron-exon junctions into relatively close proximity (Miche1 et al., 1983). 16 potential pairing EBS2-IBS2, involves a segment of the 5* exon (located 0 to 3 nucleotides distal to IBS1) that base- pairs with a sequence within the intron (EBS2). Based on the exact location of the EBS2 sequence group II introns are now classified into two subgroups - group IIA and IIB
(Jacquier et al., 1987; Michel et al., 1987). In subgroup
IIA introns, which includes all known mitochondrial group
II introns except yeast mitochondrial introns aI5g and bll
(see below) and about half of the chloroplast group II introns (Ohyama et al., 1986), the nucleotide immediately
5* to the EBS1, always pairs with the first nucleotide of the 3* exon. In addition Michel et al., (1987) propose a y-y* tertiary interaction, that joins the last nucleotide of the introns (usually a C or a U) to a nucleotide (either a G or an A) within the short single-strand region that joins domains 2 and 3.
I.B.4.b. Group II introns in yeast mitochondria
There are four group II introns in yeast mitochondria, three in the cytochrome c oxidase gene (COX I) and one in apocytochrome b (cob) gene. Intron 1 (all) and intron 2
(aI2) of the COX I gene belong to the IIA subgroup, while intron 5g of COX I (aI5g) and intron I of cob (bll) belong to the IIB subgroup. AlSg and bll are approximately the same length (0 900 bp) and do not have a coding function.
All and aI2, on the other hand, are about three times larger (0 2400 bp), are 50% homologous and contain long 17 open reading frames in phase with the preceding exon.
Genetic and biochemical analysis of all splicing defective trans-recessive mutants provides strong evidence ■ that the intron encoded ''maturase" is required for splicing in vivo
(Carignani et al., 1983).
I.B.4.C. Group II introns encode proteins related to reverse transcriptase.
Recently, Michel et al., (1985) showed that the ORFs of some mitochondrial group II introns, including all and aI2 that encode a maturase, share significant sequence homology with the reverse transcriptase genes of retroviruses. Though the significance of this homology is not clear, there is strong evidence for reverse transcription in Neurospora mitochondria (Akins et al.,
1986) and reverse transcriptase activity has been detected in the extracts prepared from Podospora anserina
(Steinhilber et al., 1985; Matsuura et al., 1986). It has, however, not been established whether this activity is associated with the Podospora mitochondrial group II introns. 18
I.B.4.d. AI5g self-splices in vitro by a novel mechanism
Group II introns are unusual in that the excised intron sequences accumulate in vivo as relatively abundant, circular RNA species (Arnberg et al„, 1980; Halbreich et al., 1980; Hensgens et al. , 1982). Since one of the products of the Tetrahymena self-splicing reaction is a circle (Kruger et al., 1982), it seemed likely that group
II introns could also self-splice. Our laboratory in collaboration with Dr. Craig Peebles at the University of
Pittsburgh reported that the last intron of the COX I gene, aI5g, self-splices in vitro by a novel mechanism (Peebles et al., 1986; van der Veen et al., 1986).
The aI5g precursor RNA self-splices efficiently when incubated in a simple salt solution consisting of 4G mM
Tris acetate (pH 7.6), 10 mM magnesium acetate and 2 mM spermidine (Peebles et al., 1986) to yield accurately ligated spliced exons and excised intron lariat, the hallmark of nuclear pre-mRNA splicing (Grabowski et al.,
1984; Ruskin et al., 1984; Padgett et al., 1984). Unlike the Tetrahymena intron, group II introns do not require a guanosine cofactor and the reaction does not benefit from the addition of monovalent cations. The reaction has a sharp temperature optimum of 45° C and is relatively insensitive to pH over a range of 6.5 - 9.0. It was subsequently shown (Schmelzer et al., 1986) that cob II, a 19 group IIB intron, also self-splices in vitro under conditions similar to those for aI5g.
Group II introns self-splice by a two step transesterification pathway (Figure 7). Cleavage at the 5’ splice-site is initiated by the nucleophilic attack of the
2*-OH group of an A residue near the 3’ end of the intron.
Subsequent cleavage at the 3* splice-site followed by exon ligation yields spliced exons and intron lariat RNA
(Peebles et al., 1987; Jarrell et al., 1988a). These products are similar to the in vitro splicing products of nuclear introns (Grabowski et al., 1984; Ruskin et al.,
1984;) and since the boundary sequences of group II introns resemble those of nuclear introns, it has been suggested that the two types of introns follow essentially the same pathway of splicing (Peebles et a l ., 1986).
I.B.4.e. Role of branch formation
In both group II and nuclear introns the first step in splicing is initiated by a nucleophilic attack of the 2’-OH group of the branchpoint nucleotide on the 5’ splice-site resulting in cleavage at the 5’ splice-site and lariat formation. In mammalian pre-mRNAs, branchpoint mutations lead to activation of nearby crytic adenosine residues
(Ruskin et al., 1985; Padgett et al., 1985), while in the yeast nuclear pre-mRNA, mutations in the TACTAAC box greatly diminish splicing (Jacquier et al., 1986b).
It is well established that in group II introns, 20 domain 6 contains the branch site. In the introns so far characterized, the branchpoint adenosine is located -7 or
- 8 nt from the 3' end of the intron (Peebles et al., 1986; van der Veen et al., 1986). Mutations of the branch point adenosine (deletion or substitution) reduced the splicing efficiency of bll dramatically (Schmelzer et al., 1987).
With aI5g, similar mutations blocked splicing under the standard splicing conditions but had little effect on the rate of the reaction at high ionic strength (van der Veen et al., 1987). These results together with the finding that trans-splicing occurs without branch formation
(Jacquier t al., 1986a) suggest that lariat formation is not strictly necessary for in vitro group II intron splicing and that accurate cleavage at the 5* splice-site can occur by site-specific hydrolysis catalyzed by water or
OH- .
I.B.4.f. Group II intron splicing resembles nuclear pre- nRNA splicing
A comparison of group I, group II, and nuclear pre- mRNA splicing mechanisms (Figure 8 ) shows striking similarities between the three mechanisms (Sharp, 1987).
While group I and group II intron splicing is essentially
RNA catalyzed, nuclear splicing is aided by trans-acting factors (proteins and snRNAs) in the splicesome. Each involves a two step pathway; the first step is cleavage at the 5’ splice-site, second is cleavage at the 3* splice- site followed by exon ligation.
The reaction intermediates and products of group II introns bear a striking resemblance to nuclear pre-mRNA splicing products suggesting that the nuclear introns are closely related to the RNA-catalyzed self-splicing reactions of group II introns. This raises the attractive possibility that splicing in nuclear introns might also be catalysed by RNA. It is possible that one or both of the two steps in the nuclear process may prove to be RNA catalyzed. Keeping in mind that the only intron sequences essential for pre-mRNA splicing are the limited consensus sequences near the 5* and 3* splice-sites (Wieringa et al.,
1983), it is highly unlikely that the catalytic RNA involved in splicing is part of the intron RNA. It is, however, possible that this hypothetical RNA catalyst is provided in trans in the form of highly conserved small nuclear RNAs (snRNAs), several of which have recently been shown to be essential for splicing of mRNA precursors in vitro (reviewed by Sharp, 1987). 22
I.B.4.g. Alternative reaction conditions
If the reaction pathway of group II introns is indeed similar to that proposed for nuclear introns (reviewed by
Green, 1986), it should involve a bi' rtite intermediate
(Figure 7) consisting of free 5* exon (5*E) and a lariat
RNA with intron and 3* exon sequences (IVS-3’E-LAK) analogous to nuclear introns. Neither of these intermediates was characterized in the initial study of group II self-splicing, indicating that the reaction is highly coupled (Peebles et al., 1986; van der Veen et al.,
1986; Schmelzer et al., 1986).
Reaction conditions were modified in order to identify splicing intermediates and products of partial reactions.
The new reaction conditions for aI5g are more efficient than the standard splicing condition (Peebles et al., 1987;
Jarrell et al. , 1988). Spermidine can be replaced by a higher concentration of magnesium (100 mM) . Surprisingly, the addition of 500 mM (NH«)2 SO« in the presence of high magnesium accelerated the reaction rate (atleast 1 0 -fold) without altering the pattern of products relative to the standard reaction. In contrast, addition of 500 mM KC1 causes a dramatic shift in the pattern of products.
Under the standard and the (NH«)2S04 reaction conditions splicing occurs via a 2 step transesterification pathway (pathway I, outlined in Figure 9). In the presence of KC1 the course of the reaction is changed somewhat 23 (pathway II) and the first step in splicing (cleavage at the 5* splice-site) occurs by hydrolysis (rather than by transesterification). The splicing intermediates (free 5* exon and the linear intron-3 'exon) carry out the second step (via transesterification) similar to pathway I.
Spliced exons, however, fail to accumalate in KC1 and are present as free 5' and 3s exon products.
Spliced exons fail to accumalate in the presence of
KC1 because of a novel reaction, termed spliced exon reopening (SER), between the excised intron and spliced exons. KC1 induces the intron to "reopen" the ligated exons, resulting in the appearance of free exons (Jarrell et al., 1988a). Bll, like aI5g, also carries out spliced exon reopening (Dietrich, Unpublished). Interestingly, however, the SER reaction is sequence specific and each excised intron reopens only its own spliced exons
(Dietrich, Unpublished).
I.B.4.h. Trans-splicing
In the case of nuclear pre-mRNA splicing, the splicing intermediates, 5' exon and intron lariat-3' exon, are held together by the "splicosome" (reviewed by Sharp, 1987), while in the case of group I introns, the internal guide sequence (IGS) serves the same function. Thus by analogy with nuclear and group I introns some type of an interaction must therefore also exist between the group II splicing intermediates, which would otherwise tend to 24 diffuse away from each other. Atleast for those group II introns that self-splice in vitro, no trans-acting proteins or RNAs can be invoked and the interaction must be direct.
Direct proof that the a5g intron recognizes its 5’ exon was provided by trans-splicing experiments (Jacquier et al., 1986). Co-incubation of 5’exon-G transcripts (5* exon plus the first nt of the intron) with seperately synthesized intron-3*exon transcripts (last 2 nt of the 5* exon plus the intron and 3* exon) results in the accurate and efficient production of spliced exons and excised linear intron without branch formation. These results suggest that there is, indeed, a strong interaction between the 5* exon and the intron.
The finding that trans-splicing occurs without branch formation provides additonal insight to the reaction mechanism and the role of lariat formation. It suggests that branch formation is not required for the first step of splicing (cleavage at the 5*splice site by nucleophilic attack of the branch site 2s -OH group) and that water or
OH- acts as the initiating nucleophile in the trans reactions . 25
I.B .4.i. Dependence on 5 * exon sequences
The role of the 5* exon in the self-splicing reaction was investigated by analysing partial deletions of the exon
(van der Veen et al., 1987; Jaccquier et al., 1987).
Transcripts containing only 2 nt of the 5’ exon were incapable of carrying out the first step of splicing
(Jacquier et al., 1986; Jarrell et al., 1987a), suggesting a requirement for more than two bases of the 5’ exon.
Interestingly, transcripts containing only the last 52 nt of the 5* exon were found to self-splice with the same kinetics as the wildtype transcript. RNAs with 35, 24 or
13 nt of the 5’ exon spliced with almost the same efficiency as the 52 nt transcript but with the appearance of a new splicing product, which turned out to be the elusive splicing intermediate, the intron-3'exon lariat
RNA. Interestingly, these 5* exon deletions <35 - 13 nt) failed to promote trans-splicing, suggesting that they lack portions of the 5’exon that form part of the exon-intron interaction essential for trans-splicing.
The intron-3'exon lariat intermediate has also recently been detected as a minor product in wild-type aI5g self-splicing reactions (Peebles et al., 1987) and provides strong support for the two step splicing mechanism
(discussed above). 26
I.B.4.j. Multiple 5*exon binding sites
Remarkably, the exon binding sites (EBS) identified by comparative sequence analysis overlapped with the'EBSl and
EBS2 identified by RNase H probing of exon-intron interactions (Jacquier et al., 1987). The existence of such interactions is suggested both by the consequences of partial deletions of the 5’ exon (see above) and by site- directed mutatagenesis (Jacquier et al., 1987). The importance of the EBS1-IBS1 pairing for the self-splicing reaction was substantiated by showing that mutations in either the EBS1 or its complementary IBS1 affected splicing in vitro whereas double mutants, in which intron-exon pairing was restored, showed normal activity.
I.B.4.k. Domain 4 is dispensible
Trans-splicing experiments that had previously been reported (Jacquier et a l . , 1988; Jarrell et al., 1988a), were basically a reconstitution of the cis reaction and did not involve branch formation. Jarrell et al., (1988b) developed an efficient trar.s-splicing system to test pairs of non-overlapping transcripts ("half molecules") interrupted within the intron. Interestingly, not all pairs of half molecules tested could trans-splice (Jarrell,
Ph.D thesis 1987). Half molecules interrupted within domain 4 are most active, yielding spliced exons and Y- branched excised intron. Further, intron domain 4 is 27 dispensible for the in vitro reaction, since a deletion of domain 4 (in trans or in cis) is with little effect on the efficiency of splicing (Jarrell et al., 1988b), suggesting that domain 4 does not play a very essential role in splicing.
I.B.4.1. Domain 5 is required for 5* exon release
Experiments designed to define essential regions of the 3' half molecule revealed the surprising result that domain 5 is required for the trans-reactions (Jarrell et al., 1988b) and that a strong interaction between domain 5 and some part of the upstream half molecule is necessary for trans-splicing. Analysis of 3* end truncations indicate that domain 5 is essential for the first step in splicing (5’ exon release) whereas 3* exon and domain 6 are not (Jarell, Ph.D thesis 1987). These data also indicate that cleavage at the 5’ splice-site does not require nucleophilic attack by the 2*-OH of the branch residue, since domain 6 is dispensible.
Jarrell et al., (1988b) showed convincingly that domain 5 is the only part of the 3' half molecule essential for initiating the first step in splicing by demonstrating that domain 5 function, when supplied as a 42 nt trans acting RNA, promotes efficient cleavage at the 5* splice- site. Supposedly, domain 5 interacts with some portion of the 5 ’ half molecule in such way as to activate the 5 * splice-site for nucleophilic attack. CHAPTER II
MATERIALS AND METHODS
11.A. Plasmid constructions
Strain ID41 - 6/161 + was the source of mitochondrial
DNA (mtDNA) for plasmid constructions,. Mitochondrial DNA was isolated as described by Hudspeth et al., (1980). The
3158 nt Hpa II - Eco RI fragment containing intron 1 of the cox I gene (see Figure 10) was purified and ligated into pBSM13+ (Stratagene, Inc.) that was cleaved with Acc I and
Eco RI to yield plasmid pSH2. The Clal-Eco RI fragment from pSH2 (see Figure 10) was subcloned into Acc I and Eco
RI cleaved pBSM13+ to yield plasmid pSH4. Plasmid pSH AC was constructed by cleaving pSH2 with Acc I and Cla I and religating the complimentary ends. Hhal-Hhal fragment from pSH2 was subcloned into AccI cleaved pBSM13+ yielding pSH
Hhal T3 and pSH Hha I T7 (in both ox-ientations).
29 28
Since group II intron splicing bears striking similarities to nuclear splicing, it has been suggested that nuclear introns may have evolved from self-splicing group II introns (Peebles et al., 1986). In the case of the nuclear introns there is considerable evidence that the
5* end of U1 snRMA is involved in recognition of 5* splice- sites, by pairing with conserved sequence GUAAGU (Zhaung et al., 1986; Parker et al., 1987). A similar situation can be envisioned for group II introns in which domain 5
interacts with the conserved sequence at the 5* splice-site and activates the 5* boundary for cleavage (Jarrell, Ph.D thesis 1987).
I.C. Dissertation Goals
1. To test whether oxi3 II, a maturase encoding group
IIA intron self-splices in vitro.
2. If the intron self-splices, to test whether the
conditions and mechanism of all are different from
those established for aI5g and bll.
3. Characterization of partial or side reactions.
4. To define regions of group II introns that are
essential for the in vitro reaction
5. Dissection of domain 5
6 . To determine whether domain 5 interacts with the
first 7 nt at the 5 ’end of the intron
7. Identify the point of interaction between domain 5
and the upstream half molecule. 30
II.B. Plasmid preparation
Plasmid DNA was prepared from 125 ml of YT containing ampicillin (0.05g/ml) as described in the GemSeq Technical manual (Promega Biotech, Madison, Wis.). Miniprep plasmid
DNA was prepared by the alkaline lysis method (Maniatis et al., 1982).
II.C. Transcription and Purification of RNA
RNA was transcribed and purified as described by
Jarrell et al., (1988a). Unless otherwise indicated, the plasmids used to synthesize all precursor RNA were linearized in the downstream polylinker with Eco RI and transcribed using T3 RNA polymerase. While plasmids used to synthesize aI5g RNA were linearized with Hind III and transcribed using T7 RNA polymerase.
II.D. All Splicing reactions
RNA was diluted in water and mixed with an equal volume of buffer stock solution to achieve a final concentration of 40 mM Tris-HCl, 100 mM MgCl2 , 1M NH*Cl and
0.1% SDS. Reactions were incubated at 40°C for 2 hours.
Samples were then ethanol precipitated, the pellet vacuum dried and dissolved in 10 ul of water. An equal volume of loading buffer was added and samples loaded directly onto
3.5% polyacrylamide/8 M urea gels (40:1 acrylamide-bis- acrylamide).
AI5g splicing reactions were done as described by 31 Jarrell et al., (1988a).
II.E. Site-directed Mutagenesis
Oligonucleotide-directed mutagenesis was carried out essentially as described by Kunkel et al., (1986). Single strand DNA was obtained from Plasmid pJDl (Jarrell et al.,
1988a) transformed into E. coli CJ236 (dut~ ung"). The oligonucleotide was annealed to the DNA by heating to 90°C, then slow cooling to room temperature. The second strand was synthesized by initiating the polymerase-ligase reaction and incubating at 37° C for 90 min. Dilutions of this reaction were transformed into competent E. coli DH5
F ’ . Transformants were initially screened by restriction site analysis and later confirmed by dideoxy sequencing of double-stranded plasmid DNA. E. coli CJ236 and enzymes were purchased from Bio Rad Laboratories.
II.F. Purification and end-labeling of oligonucleotides
Oligonucleotides were synthesized on an Applied
Biosystems DNA synthesizer, using the 0.1 umol fast cycle set for automatic removal of the trityl group. The oligonucleotides were removed from the synthesizer and incubated at 55°C for 6-12 hours. Samples were lyophilized 3-4 times and then passed over a G25 spun column. The oligonucleotides were denatured by heating (10 min at 70°C, 5 min on ice) before end-labeling using T4 polynucleotide kinase and y32? ATP (Maniatis et al., 1982). 32
II.G. RNA sequencing
The spliced exons were sequenced using reverse transcriptase by the dideoxynucleotide method as described by Lane et al. , (1985), except that the oligonucleotide used for sequencing was end-labeled.
II.H. Northern Blots
In vitro splicing reactions were fractionated on 3.5% polyacrylamide gels, immobilized onto nitrocellulose
(Genescreen) and filters were probed with 5*-end labeled oligonucleotides. Hybridiztion temperatures were calculated independently for each oligonucleotide using the formula Th = 2(# A+T) + 4(#G+C).
II.I. 3* end-labeling of RNA
RNA (2-3 ugm) was extracted with phenol-chloroform- isoamyl alcohol, passed over a G50 spun column and ethanol precipitated. The RNA pellet was vacuum dried and brought up in 5.0 ul. The RNA was 3’ end-labeled with 5"3 ap[pCp] using T4 RNA ligase as described by England et al., (1980).
II.J. Limit T1 digest
Unlabeled all precursor RNA was reacted in the splicing buffer, ethanol precipitated and the RNA 3* end- labeled (described above). The RNA was then fractionated on a 3.5% polyacrylamide/8 M urea gel (40:1, acrylamide to bis acrylamide). Gel purified IVS-LAR and IVS-LIN RNAs were 33 brought up in 10 ul of T1 digestion buffer <10 mM tris-HCl,
5 mM EDTA and 300 mM NaCl, pH 7.5). 1 ul of T1 was added and samples incubated at 30°C for 1 hour. Reactions were stopped by adding an equal volume of gel loading buffer and the products were fractionated on a 25 % polyacrylamide, 8
M urea gel. An oligo (dT) ladder (4 to 22 nt long), 5* end-labeled with [yaaP]-ATP and T4 polynucleotide kinase, was used as a size standard.
II.K. Other Methods
Debranching experiments and primer extension analysis were done as described by Peebles et al., (1986).
II.L. Enzyme reagents
Restriction enzymes were purchased from Bethesda
Research Laboratories and New England Biolabs. Phage T4
DNA ligase was purchased from New England Biolabs. T3 RNA polymerase was purchased from Stratagene, Inc, while T7 RNA polymerase and T4 polynucleotide kinase was purchased from
United States Biochemicals. Ribonuclease T1 and phage T4
RNA ligase were purchased from Pharmacia. Reverse transcriptase was purchased from Seikagaku America Inc. CHAPTER III
RESULTS
III.A. Teat of the generality of group II self-splicing
Based on several highly conserved base pair interactions, group II introns are now classified into two subgroups - group IIA and IIB (See Chapter I and Michel et al., 1987). There are four group II introns in yeast mitochondria, three in the cytochrome c oxidase subunit I
(cox I) gene and one in the apocytochrome b (cob) gene.
Recent studies in several laboratories have shown that both introns belonging to the IIB subgroup (cox I intron 5g
(aI5g) and cob intron I (bll) self-splice in vitro (Peebles et al., 1986; Van der Veen et al., 1986; Schmelzer et al.,
1986).
In order to test whether self-splicing is a general feature of group II introns, other introns needed to be examined. The remaining two introns, of the IIA subgroup
(cox I intron 1 (all) and intron 2 (aI2), are about three times larger in size (2.4 Kb vs. 0.9 Kb) and contain long open reading frames (ORFs) known to code for maturases.
Strong genetic and biochemical evidence shows that the maturase encoded by all is required for its splicing in 34 35 vivo (Carignani et al., 1983). Our lab has confirmed these data for all using independently isolated mutants
(Mecklenberg, 1986).
This section deals with experiments done to test whether all could self-splice in vitro. There were two possibilities: first, it was possible that the intron would
self-splice efficiently in vitro in the complete absence of
proteins; second, it was possible that the intron would be
so dependent on its maturase that no splicing would be detected in vitro, in which case, the intron would prove to be a very suitable substrate for extract dependent in vitro
studies.'
III.A.l. Cloning of intron 1 of the COX I gene
To test whether all self-splices in vitro, I
constructed plasmid pSH2 (Figure 10). A Hpa II - Eco RI
fragment of yeast mtDNA, containing all and flanking exon
sequences, was cloned behind the T3 promoter of pBSM13+
(see Methods). Transcription of pSH2 following
linearization with Eco RI (using T3 RNA polymerase)
generates a 3158 nucleotide (nt) precursor RNA spanning the
insert. That in vitro precursor mRNA analog contains a 467 nt 5* exon (comprised of 27 nt of vector sequence, 270 nt of cox I gene untranslated leader sequence and all of exon
1), the entire intron 1 (2448 nt), and a 290 nt 3' exon
(comprised of the entire 36 nt exon 2 coding sequence fused to 254 nt of intron 2). Plasmid pSH2 has been linearized 36 at Eco RI in all the following experiments, unless indicated otherwise.
III.B. Features of the all self-splicing reaction
III.B.l. All RNA is inactive in the standard aI5g splicing buffer
Initial investigation of the in vitro reactivity of this model pre-mRNA surveyed the reaction conditions defined for aI5g by Peebles et al,, (1986) and Jarrell et al., (1988a). Incubation of the run-off precursor RNA under conditions normally used for splicing of group IIB introns (2mM spermidine, lOmM Mg 2 + , 40mM Tris-acetate, pH
7.6 and an incubation temperature of 45°C) revealed no i splicing activity, as shown in Figure 11, lane 2. In the presence of lOOmM MgClz, spermidine is neither stimulatory nor essential for the aI5g splicing reaction (Jarrell et al. , 1988a). While the aI5g self-splicing reaction requires at least 5mM MgClz and proceeds at similar rates throughout the range of 10-100 mM magnesium (Peebles et al., 1986), all RNA remains inactive even in the presence of 100 mM Mg2 + (Fig 11, lane 3). 37
III.B.2. All has an absolute requirement for monovalent cations
Next, the reactivity of the RNA was examined in the presence of 100 mM Mg 2 + supplemented with 5uO mM (NHihSC^,
NH«C1 or KC1. These conditions enhance the overall reaction rate of aI5g and some of these also dramatically alter the pattern of products (Jarrell et al., 1988a). As shown in Figure 11, lanes 4-6, the all precursor RNA is very reactive in the presence of monovalent cations and undergoes a magnesium dependent rearrangement reaction leading to the appearance of reaction products. Thus, the all RNA, unlike aI5g, appears to have an absolute requirement for monovalent cations.
While 0.5 M NH«C1 stimulates the all reaction, an equivalent amount of KC1 yields a radically different array of products (Figure 11, lanes 5 and 6 ). Additional experiments examining salt effects in the presence of 1 0 0 mM MgCl2 showed that the addition of 0.5 M LiCl or NaCl
(Figure 12, lanes 2 and 4) did not support any reaction.
Substitution of chloride ion by acetate ion gave similar results (lanes 6 - 8 ). Surprisingly, the RNA is only weakly reactive in the presence of 0.5 M (NH«)2 SCU (lane 9) which is the most reactive condition for aI5g (Jarrell et al. , 1988a).
In a separate experiment (not shown) I determined that 38 the same reaction occurs throughout the range of pH values between 6.5 and 8.5. Tris chloride (40 mM) buffer at pH
7.5 was, therefore, selected for subsequent experiments.
Other buffering agents are interchangeable for a!5g reactions but this point was not tested for all.
III.B.3. All RNA has a higher threshold for magnesium than al5g
Since the precursor RNA undergoes a magnesium dependent rearrangement reaction in the presence of NH«C1, the response of the reaction to varying concentrations of magnesium ion was examined in the presence 0.5 M NH*C1
(Figure 13). While the RNA remains essentially inactive at concentrations of 10 and 25 mM (lanes 2 & 3), reaction products are weakly detectable at 50 mM (lane 4). The RNA is, however, most reactive in the presence of 100 mM magnesium (lane 5), giving rise to reaction products.
While aI5g is reactive in the range of 10 - 100 mM magnesium, all RNA in contrast, is almost inactive below
100 mM and appears to have a higher threshold for magnesium. 39
III.B.4. NH«C1 as the standard splicing buffer
The number of products obtained in the presence of added (NH«)2 S0 « was the smallest while the most complex pattern was obtained with added KC1. There is a set of products that is common to all three reactions and another set present only in the KC1 reaction. The reaction condition containing NH«C1 was selected as the standard because the array of products appeared to be intermediate between the two extremes noted above.
Having selected NH«C1 as the standard splicing condition, I examined the effect of varying the NH 4 CI concentration while keeping the concentration of MgClz constant at 100 mM (Figure 14). At concentrations of 50 and 100 mM (lanes 2 and 3 respectively), the RNA was only partially reactive and failed to yield the complete set of reaction products. While the reaction proceeds weakly at
250 mM NHtCl (lane 4) and fairly well at 500 mM (lane 5), a concentration of 1 M appears to permit the maximum yeild of products (lane 6 ). Though the products of the reaction are essentially the same at 2 M NH4 CI, they occur in varying proportions (lane 7). It is interesting to note that the all reaction requires 1 M NH«Cl for maximal activity, 2 fold higher than the salt requirement for aI5g. 40
III.B.5. All reactions have a temperature optimum of 40*C
The effect of incubation temperature on the in vitro
reaction is shown in Figure 15. The RNA is optimally
reactive at 40°C in the presence of 100 mM Mg** and 1 M
NH«C1 (lane 6 ). As the temperature is lowered to 20° C
(lanes 2 - 5) the reaction rate declines rapidly and
reaction products are barely detectable. At 45°C (lane 7) or above (not shown) considerable non-specific degradation of the RNA is observed. Interestingly, all has a
temperature optimum of 40°C, 5°C lower than the sharp optimum of 45°C reported for aI5g (Peebles et al. , 1986).
An incubation temperature of 40°C was, therefore, chosen for all of the following experiments.
III.B.6 . The reaction is unimolecular and time dependent
A time course of the NH4 CI reaction (Figure 16) shows that the precursor is quite reactive in 1 M NH4 CI, products being readily detectable within 1 0 minutes of incubation
(lane 2). Samples incubated for 30, 60 and 120 minutes
(lanes 3 - 5) indicate that the reaction is progressive over time with most of the precursor (about 80%) being converted to products in 120 minutes. Clearly a single set of products is obtained in all the samples — suggesting that they are all products of a primary rather than a secondary reaction.
The above results indicate that the all pre-mRNA 41 analog undergoes a time dependent rearrangement reaction.
If the reaction is truly unimolecular, the relative rate of the reaction should not respond to changes in reactant concentration. A bimolecular (or higher ordered) reaction, on the other hand, should exhibit accelerated relative rate with increased reactant concentration. Comparison of the reaction time courses for two different concentrations
(that differed by 20-fold) of the all precursor RNA showed similar relative rates of accumulation of products and no apparent shift in product distribution (not shown). This result is consistent with our interpretation that the products result from a unimolecular (intramolecular) reaction involving the precursor RNA.
III.B.7. Optimum reaction conditions
From the above studies of reaction conditions, the standard condition for in vitro self-splicing of all was established as 100 mM MgCl2 , 1M NH«C1, 40 mM Tris-chloride
(pH 7.5) and an incubation temperature of 40°C. The aI5g self-splicing reaction, in contrast, requires a sharp temperature optimum of 45°C and is stimulated maximally by
(NH«)2 S0 « at 0.5 M; the other parameters being the same for the two introns. For each intron under its optimal reaction condition, comparable extents of reaction are obtained --- most of the precursor is converted to products in 1 - 2 hours of incubation (compare the all standard reaction with Jarrell et al., 1988a). 42
III.C. Characterization of the NH«C1 reaction products
On the basis of their mobilities relative to size standards (not shown), it was possible to define the NH 467 nt and the 3* exon (3’E) 290 nts. If accurate splicing should occur in vitro it would yield spliced exons (5’E- 3*E) 757 nt in length; there is a product roughly that size. The RNA species migrating slower than precursor RNA was tentatively identified as IVS-LAR (excised intron lariat) and the two RNA species migrating somewhat faster than precursor RNA, as excised linear intron (IVS-L) and the intron-3'exon RNA (IVS-3’E) (see Figure 16). Since the IVS-LAR of all is much larger than aI5g (2.4 Kb vs. 0.9 Kb) the gel conditions were modified so as to enable the large IVS-LAR RNA to enter the gel. A 3.5% polyacrylamide/ 8 M urea gel, with an acrylamide-to-bis-acrylamide ratio of 39:1 was used for electrophoresis in all the subsequent all experiments. AI5g reaction products, in contrast, are typically fractionated on 4% polyacrylamide/ 8 M urea gels, containing acrylamide and bis-acrylamide at a ratio of 29:1. 43 III.C.l. Identification of products containing the 3'exon Reaction products containing the 3*-end of the precursor RNA undergo a mobility shift when the template DNA is linearized at a different restriction site. To identify products retaining the 3*exon, experiments were done using precursor RNA with a 150 nt 3’ extension (using as template plasmid pSH2 linearized at the vector Pvu II site, see Figure 10). A comparison of splicing products derived from the Eco RI and Pvu II linearized precursor RNA is shown in Figure 17, lanes 2 and 3. The smallest RNA (757 nt) that shifted to slower mobility in this experiment was the band identified as 5’E-3*E. While this also holds for the precursor RNA and the IVS-3’E RNA (lane 3) there was, however, no RNA the size of free 3’E (290 nt) that underwent a mobility shift. Additional experiments were done to identify products containing the 3*-end of the precursor RNA. Transcripts were 3*-end-labeled (see Materials and Methods) using T4 RNA ligase and 5'3l2 p[pCp] as described by England et al., (1980). The RNA was then incubated in the standard splicing buffer for 60 min. IVS-3’E (reaction intermediate containing the intron and the 3’E) which is 467 nt smaller than the precursor RNA, is the only intron-containing product that remains labeled (Figure 18, lane 4). While 5’E-3’E RNA is also labeled there is no indication of any 44 product containing only 3* exon sequences. This experiment thus validates the earlier observation that free 3*E is not a reaction product. In contrast, aI5g in the presence of NH4 CI yields in addition to the standard splicing products, free 3*E and 5*E. III.C.2. First test of the validity of the assignments Products containing exon sequences were first identified by probing northern blots of unlabeled reacted RNA with exon-specific oligonucleotides. The synthetic oligonucleotides complementary to the exon and intron sequences were end-labeled and used as probes in northern blot analysis (see Materials and Methods). Products identified as 5*exon (5’E) and spliced exons (S’E-S’E) in Figure 19, lane 4; hybridize to the 5*exon-specific probe. The RNA identified as the spliced exons also hybridizes to the 3*exon probe (lane 2). Neither 5*E nor 5*E-3’E hybridizes to the intron-specific oligonucleotide (lane 6 ). RNA (randomly labeled at uracil residues) in lanes 1, 3 and 5 is used as a marker. The signals from the larger RNA products are ambiguous because of technical difficulties in transferring such large RNAs from 3.5% polyacrylamide gels onto the nitrocellulose filters. The identification of the IVS-LIN RNA will be dealt with in detail in a later section. Northern blot data are consistent with the tentative assignments made for 5'E and 5’E-3'E. The data indicate 45 that 5'E and S’E-S’E contain at least that region of the 5* and 3* exon sequence complementary to the oligonucleotide used as a probe. The 5* ends of these RNAs were mapped by primer extension using the same oligonucleotide (see Materials and Methods). Both 5'E and 5’E-3’E were gel purified and used in primer extension experiments to map their 5* ends. Primer extension using a 18 nt primer (complementary to a sequence in 3*E) with the 757 nt RNA (5*E-3'E) yields, as expected, a major product that is approximately 485 nt (Figure 20, lane 3). Primer extension using a 21 nt long oligonucleotide (5* exon-specific) with the 467 nt RNA (free 5*E) gave a major product (lane 5) that is roughly the expected size (359 nt). III.C.3. Accurate ligation of exons Evidence from the last few experiments agrees with our initial assesment that the 757 nt RNA species is 5'E-3*E. It was, however, necessary that it be proven unequivocally by sequencing across the splice junction. Sequencing was done by the Sanger dideoxy chain termination method using reverse transcriptase. An end-labeled 3*exon-specific primer was annealed to purified 5E-3*E and extended using reverse transcriptase in the presence of unlabeled dNTPs and ddNTPs (see Methods). The cDNA sequence, 5* ACTAAAA / CATTAAA 3* (Figure 21), confirms the identification of 5’E- 3'E and shows that it is the product of accurate ligation of 5* exon to 3*exon. 46 III.C.4. The slowly migrating RNA species is excised intron lariat (IVS-LAR) The product labeled IVS-LAR was identified as excised intron lariat by the results of several experiments. First, the electrophoretic mobility of IVS-LAR varied relative to the other (linear) RNAs in the sample, when the cross-linking ratio of the polyacrylamide gel matrix was varied from 19:1 to 39:1 (not shown). This is characteristic of a molecule with topological constraint (non-linear RNA), migrating much more slowly than the precursor RNA. In the second experiment, unlabeled precursor RNA was reacted in 1 M NH4 CI, samples ethanol precipitated and brought up in 10 ul. The reaction products were then 3* end-labeled using 5’3 2 p[pCp] and T4 RNA ligase and analyzed on a 3.5% polyacrylamide gel (Figure 22, lane 2). As a control, uniformly labeled transcript was reacted in NH4 CI (lane 1). Each one of the reaction products was 3* end- labeled (lane 2 ), indicating that each has a free 3' - hydroxyl end. Since the product labeled IVS-LAR is also end-labeled, it is probably a lariat RNA becauce circular RNA molecules do not have 3*-hydroxyl ends. Additional support was provided by the results of the third experiment shown in Figure 23. IVS-LAR RNA purified from preparative acrylamide gels was incubated without Al (lane 2) or with a debranching extract (lane 3). Extracts i of HeLa cells contain a debranching activity that hydrolyzes 2 ’ - 5' phosphodiester bonds of RNA lariats, converting them to linear molecules (Ruskin et al., 1985). As a positive control the aI5g IVS-LAR is shown to be debranched by the same extract (lane 6 ). The all IVS-LAR is converted to a faster migrating RNA (lane 3) that comigrates with IVS-LIN RNA. Given the known specificity of the extract used, the all IVS-LAR junction appears to be a 2* - 5* phosphodiester bond similar to that found in excised nuclear introns and the other group IIB introns studied. III.C.5. Accurate 5* Cleavage It was necessary to map precisely, the 5’ ends of the IVS-LAR and IVS-BL (consisting of both linear intron and broken form of the intron lariat) to determine whether the RNAs were accurately cleaved at the 5* splice site. The same 2 2 nt intron-specif ic oligonucleotide used in the northern blots, complementary to the region 170 - 192 nt from the 5* end of the linear intron, was 5* end-labeled, annealed to purified IVS-BL and IVS-LAR, and extended using reverse transcriptase and unlabeled dNTPs. The length of the extended product was measured relative to a DNA sequencing ladder. Both IVS-LAR and IVS-BL yield the same 192 nt extension product (Figure 24, lanes 1 and 2 respectively). The stop site maps to the first nucleotide 48 of the intron and the product is of the expected length if cleavage occurs at the 5* splice site. This result shows that the 5* ends of the IVS-LAR and IVS-BL result from accurate cleavage at the 5* splice junction. In addition the strong stop to reverse transcription with the IVS-LAR provides further evidence that it is indeed a lariat RNA. III.C.6 . Technical problems in mapping the branch point It is not possible to map the branch point of group II intron lariat RNAs by a primer extension experiment (as is usually done for nuclear introns) because the 3* extensions are only 7-8 nt in length. Since all IVS-LAR is difficult to recover intact from gels due to its large size (2448 nt), I was unable to obtain sufficient quantities of the IVS-LAR to map its branch point. The results of a different experiment solved this problem. III.C.7. Summary and implications of product characterization The data presented above clearly establish that all self-splices efficiently in vitro to yield accurately spliced exons and excised intron lariat. I have identified the major reaction products (5’E, IVS-BL, IVS-LAR, 5'E-3’E) and mapped their 5* ends and confirmed that the spliced exons are accurately ligated. In addition, I have shown that a group IIA intron (all) self-splices under conditions slightly different from those of the two group IIB introns 49 (al5g and bll). Specifically, unlike aI5g and bll, monovalent cations are absolutely essential for all self splicing. The reaction has a higher threshold for magnesium and the salt requirement is two-fold higher for all. It has a broader temperature optimum of 37 - 40°C as compared to sharp optimum of 45®C for aI5g and bll. Jarrell et al., (1988a) have proposed two reaction pathways that summarize the sequential steps for splicing of aI5g (Figure 9). While pathway I (transesterification) predominates under low salt "standard conditions" and is accelerated about 10-fold in the presence of 100 mM MgClz and 500 mM (NHOzSO*, two-thirds of the reacting molecules carry out the alternative first reaction of pathway II (hydrolysis) when 500 mM KC1 is provided. With all, however, it appears as if half of the reacting molecules in the presence of 100 mM MgCl2 and 1M NH*C1 undergo pathway I and the other half pathway II, accounting for the major reaction products. Thus, even though there is some branching in NH*C1 much of the intron is present as IVS-L and IVS-3*E (see Discussion). III.D. All yields some novel reaction products in KC1 Initial experiments examining salt effects on the in vitro reaction showed that the all precursor RNA in the presence of KC1 yields the most extreme deviation from the pattern of products obtained in the presence of NH«C1 (Figure 11, lane 6 ). Interestingly, in addition to the set 50 of products (5‘E, 5*E-3'E, IVS-3'E and IVS-LIN) common to the two conditions, the precursor RNA in KC1 yields novel linear and non-linear products. The IVS-LAR RNA prominent in the NH4C1 reaction, is noticeably absent from the KC1 reaction and the IVS-3*E RNA is barely detectable. The splicing reaction in KC1 was further investigated in order to identify the new reaction products. Ill.D.1. Further analysis of KC1 effects on the all reaction Ir the following experiment the KC1 concentration in the splicing buffer was varied while all other parameters were set at the optimal standard condition (Figure 25). The RNA is weakly reactive in the presence of 50 and 100 mM KC1 (lanes 1 & 2 respectively). It is interesting to note that the products there are essentially the same as in the standard reaction except for the absence of the IVS-LAR. Higher concentrations of KC1 (250 mM in lane 3, and 500 mM in lane 4) result in the appearance of the novel linear and non-linear RNAs. The precursor, however, appears to be maximally reactive at 1 M KC1 (lane 5). A time course of the KC1 reaction (Figure 26) shows that the precursor is very reactive, products being clearly visible after only 5 minutes of incubation. The novel reaction products begin to appear after 2 0 minutes of incubation and increase progressively with time. Over 80% of the precursor is converted to products in 1 2 0 51 minutes. Interestingly, the novel linear and the novel, non-linear reaction products appear at the same point in the time course suggesting that they may be products of a secondary reaction. 1II.D.2. Characterization of novel EC1 products The identities of the novel linear and non-linear reaction products were established by several experiments. First, Northern blot analysis (Figure 19, lane 6 ) showed that the 520 nt novel linear product hybridizes with the intron probe (complementary to the region 170 - 192 nt from the 5 ’end of the intron) and thus appears to be a 5* intron fragment. The smaller intron fragment (262 nt) was not detected in this analysis because it was run off the gel in an attempt at getting better resolution of the larger products. The identity of the smaller novel product was established by the following experiment. If the two linear products in KC1 are indeed intron fragments that contain the 5* end of the intron, primer extension using the 22 nt intron-specific primer should yield a cDNA product that is 192 nt in length. The intron-specific oligonucleotide was end-labeled, annealed to the gel purified intron fragments and extended using reverse transcriptase and dNTPs. The 5’ end of the extension product was mapped precisely, to the nucleotide, by running them alongside a sequencing ladder (Figure 27). A single 192 nt primer extension product was 52 obtained with both the intron fragments (lanes 1 & 2 ). Thus, the two fragments have the same 5* but different 3* ends. Based on the size of the fragments the 3* cleavage sites within the intron can be approximated (520 nt and 262 nt from the 5* end of the intron). The 3’ ends were, however, not mapped precisely. It is already established that IVS-LAR RNA converts to a linear form on breakage and migrates like IVS-LIN RNA (Peebles et al., 1986). In an attempt to identify the non linear RNA species, they were purified from preparative gels and fractionated next to a control sample containing KC1 reaction products (Figure 28, lanes 4 and 5). The non linear RNA species give rise to two linear RNAs that co- migrate with the larger novel linear reaction products of the KC1 reaction (marked by arrows). These linear RNA products, which are absent in NH4 CI, migrate faster than full-length IVS-LIN RNA. This suggested that those non linear RNA species are either circular or lariat molecules that lack part of the intron. In order to determine whether the non-linear RNAs are circular or lariat molecules, unlabeled precursor RNA was reacted in 1M KC1 and the products 3' end-labeled with 5>32p[pCp] and T4 RNA ligase. While circular RNAs lack a 3*OH group, lariat RNAs have short tails with a free 3’ OH group that can be end-labeled. As shown in Figure 29 (lane 2), the two non-linear RNAs were readily end-labeled, 53 indicating that each has a free 3*-hydroxyl end and is, therefore, a lariat rather than a circle. The susceptibility of the smaller IVS-LARs to the debranching extract was examined (not shown). Surprisingly, the IVS-LARs in KC1 did not debranch. I have tested the specificity of the debranching extract by using different substrate RNAs (see section III.I.3.a.) and found that the debranching activity appears to be specific for a G - A branch. It is, therefore, likely that the IVS-LARs in KC1 have a different branch point. 54 III.D.3 A proposed pathway for the KC1 reaction The evidence presented so far suggests that the novel KCl products (5* intron fragments, the small IVS-LARs) result from post-splicing reactions involving the linear excised intron. Figure 30, outlines the most likely splicing pathway in KCl. In the first step, 5* cleavage takes place by hydrolysis rather than transesterification, releasing the free 5' exon and the linear IVS-3’E. The second step is attack by the 3*-OH group of the released 5' exon at the 3* splice junction followed by exon ligation, to yield spliced exons and excised full-length linear intron. Like the Tetrahymena rRNA intron (Zaug et al., 1983), the excised linear intron is capable of undergoing further reactions in KCl (Figure 31). The intron cleaves itself at either of two sites near its 5* end yielding 5* intron fragments and IVS-LARs smaller than the full-length IVS- LAR. To test this directly IVS-LIN was purified and re incubated in KCl. It was extremely difficult to obtain IVS-LIN RNA, free of contaminating precursor and IVS-3*E RNAs, because of the proximity of those RNAs. The linear intron when incubated in KCl (Figure 32, lane 3), undergoes a post-splicing reaction to yield products that co-migrate with the novel IVS-LARs and the intron fragment from the KCl reaction (lane 5). The smaller intron fragment was, however, mistakenly run-off the gel. This result clearly 55 supports my model and shows that the all intron undergoes post-splicing reactions to yield IVS-LARs lacking portions of the intron. The post-splicing reaction is, however, a very inefficient reaction but the fact that it occurs efficiently during the course of the in vitro reaction suggests that the presence of some other RNA (e.g., exon sequences) may enhance the rate. III.E. Spliced exon reopening (SER) IXI.E.l. All does not reopen spliced exons Under KCl reaction conditions both aI5g and bll yield a significant amount of free 3’E RNA (plus free 5*E RNA). It was shown that most of that product results from a post splicing reaction between excised intron and spliced exons that was termed "spliced exon reopening" (or SER) by Jarrell et al., (1988a). The SER reaction carried out by al5g is most evident under the KCl reaction condition but is also quite evident in NH«C1. The free 5*E present in reacted samples of the the all precursor RNA, on the other hand, is probably a reaction intermediate since IVS-3’E is also present and there is no obvious accumulation of free 3*E RNA. Since all does not yield free 3*E even under KCl conditions, it appears that all does not carry out the SER reaction. This was directly tested in the following experiment. To obtain purified spliced exons, uniformly labeled precursor RNAs, for all or aI5g, were reacted in the 56 presence of 1M NH«C1 or 0.5 M (NHi^SO* respectively, for 1 hour to allow spliced exons to accumulate. The RNAs were size fractionated by gel electrophoresis, the band corresponding to the spliced exons excised and the RNA eluted. The spliced exons were then incubated either with or without an excess (30 fold) of each unlabeled precursor under KCl conditions (Figure 33). Both the all and aI5g spliced exon RNA were stable and no free exons were observed when incubated for 1 hour in a buffer containing 100 mM MgCl2 plus 0.5 M KCl (lanes 2 and 5 respectively). The addition of an excess of aI5g unlabeled precursor RNA to its spliced exons prior to incubation results in the appearance of free exons (lane 6). The all spliced exon RNA, however, remains stable and does not yield free exons even after the additon of an excess of its unlabeled precursor (lane 3). This result implies that all does not carry out the SER reaction and is consistent with our interpretation of the reason for the absence of free 3* exon among the reaction products of the forward reaction. 57 III.B.2. SER is sequence specific To test whether the ability of aI5g to do SER is intron specific, a heterologous SER reaction was done (Figure 34). Interestingly, while aI5g carries out SER efficiently on its own spliced exons (lane 6) it was unable to reopen all spliced exons (lane 7). All RNA, however did not carry out SER on its own (lane 3) or on aI5g spliced exons (lane 4). This suggests that the reaction may be sequence specfic. It was subsequently shown by Jarrell (1987) that both forms of excised aI5g RNA (IVS-BL and IVS- LAR) catalyze the SER reaction. Craig Peebles lab is currently investigating which sequences in spliced exon RNA are required for SER. Since aI5g and all belong to differentsubgroups it was essential to ask whether bll, which belongs to the same subgroup IIB as aI5g, could carry out SER. R. Dietrich (unpublished studies) clearly demonstrated that bll, like aI5g, catalyzes the SER reaction. The bll activity was tested in a heterologous experiment with aI5g (similar to the one described above). Both heterologous reactions yielded no products consistent with the earlier finding that the SER reaction is sequence specific. The ability to carry out SER, therefore, is not limited to just one intron but may be a subgroup specific feature. 58 III.F. Much of the intron OBF can he deleted One of the major goals of this dissertation was to test if all, a maturase encoding group IIA intron, could self-splice in vitro. My work with all is the first .demonstration of a maturase encoding intron of either group I or group II shown to self-splice in vitro. All is also the first example of a self-splicing group IIA intron, suggesting that the ability to self-splice maybe an inherent property of group II introns. If the right conditions are found, all group II introns may be capable of self-splicing in vitro. Having accomplished the first goal of this dissertation, my next goal was to probe the function(s) of group II intron domains. All self-splicing has greatly benefited from parallel experimental approaches undertaken with aI5g splicing. It has recently been demonstrated (Jarrell et al., 1988b) that all of intron domain 4 of aI5g (which does not contain an ORF) can be deleted without any effect on the in vitro splicing reaction. Domain 4 is highly variable in length (25 nt in bll and 1870 nt in all) and appears to be a non-essential region of group II introns; nonetheless, it is conserved. In every case where a group II intron contains an ORF, most of it is "inserted” in domain 4 (Michel et al., 1983). Since I have shown that the maturase encoded by all is not necessary for self splicing in vitro, it is worth considering whether regions 59 of the RNA corresponding to the ORF may be dispensible under self-splicing conditions. This was directly tested by constructing a shortened version of the all intron. Sixty percent of domain 4 was deleted by cleaving plasmid pSH2 at its unique Acc I and Cla I sites (see Figure 10) and religating the complementary ends to form plasmid pSHAAc. When cleaved with EcoRI and transcribed with T3 RNA polymerase, a precursor RNA containing less than half of domain 4 was obtained. As shown in Figure 35, RNA lacking domain 4 was as reactive as the control substrate (Compare lanes 10-12 with lanes 4-6) yielding reaction products analogous to those obtained with the full-length intron. The identity of spliced exons was confirmed by sequencing across the splice junction. RNA corresponding to spliced exons was purified, annealed to an end-labeled oligonucleotide, extended using reverse transcriptase, dNTPs and ddNTPs. The sequence across the splice junction was the same (not shown) as that obtained for the accurately spliced exons from the full-length precursor (Figure 21). The product that migrates slower than the deleted precursor was confirmed to be excised intron lariat by a debranching experiment (shown in the next section). The fact that the shortened intron form of all is as reactive as the full-length intron supports the finding by Jarrell et al., (1988b) that intron domain 4 is not essential for the in vitro reaction. It is possible 60 that domain 4 plays a role in vivo; however, some feature of the in vitro reaction has rendered it non-essential. III.F.l. Location of the branch site in the shortened IVS- LAR An interesting feature of group II introns is that domain 6 contains a highly conserved unpaired adenosine residue, the 2'-OH group of which acts as the attacking nucleophile in the first step of splicing. Of the two group IIB introns characterized so far (aI5g and bll), the branch point adenosine is the 8*6 nucleotide from the 3* end of intron (Van der veen et al., 1986 and Schmelzer et al., 1986). As pointed out earlier, characterization and identification of the all branch point was greatly hindered because of its large size (2448 nt). This problem was alleviated by using the shortened intron form of all. The IVS-LAR (shortened intron form) was gel purified, incubated in the absence (Figure 36, lane 5) or in the presence of the debranching extract (lane 6). Treatment of the purified RNA with the debranching extract (lane 6) converted most of the slowly migrating RNA to a faster migrating RNA that comigrates with the product that is most likely the IVS-BL while samples not incubated in the debranching extract remain unaffected (lane 5). As a positive control aI5g IVS-LAR is shown to be debranched in lane 3. Given the known specificity of the debranching extract it appears as if the shortened intron lariat has 61 the same kind of branch point as the full-length intron lariat. As shown above, the shortened intron lariat is an abundant product compared to the full-length intron lariat under NH To determine whether the branch site is within that T1 fragment, unlabeled shortened precursor SNA was reacted in 1M NHiCl, the products 3' end labeled with 5*3*p[pCp] and RNA ligase, fractionated on a polyacrylamide gel and products IVS-LAR and IVS-BL extracted from gel slices. Those RNAs were digested to completion with RNAse T1 and the products analysed on a 25% polyacrylamide gel with 5* end-labeled oligodeoxyribonucleotides as rough size standards (Figure 37). IVS-BL yields mostly a fragment migrating with the expected mobility (9-mer) and a small amount of a second fragment with slower mobility (lane 3). IVS-LAR RNA, on the other hand, yields a single fragment migrating like the minor product from IVS-BL (lane 2). These data suggest that most of IVS-BL of all, like aI5g, 62 is unbranched. Due to the presence of the branch, the terminal T1 fragment from the IVS-LAR migrates slower than the 9 nt T1 fragment of IVS-BL. While this experiment does not locate the precise branch point it confirms the expectation that it is present in the last 9 nt of the intron (near or at the predicted site). It should be noted that the adenosine residue at position -7 is the only A (adenosine) in that region of domain 6. III.G. Studies using portions of all II1.G.1. Trans-splicing Jarrell et al., (1988b) recently demonstrated that "half molecules" of aI5g interrupted within or deleted for part of domain 4 trans-splice in vitro. To test whether all can trans-splice, I took advantage of three unique restriction sites (Seal, AccI and Clal) within domain 4 of all (see Figure 38) to prepare "half molecules". Randomly labeled runoff transcripts were synthesized (using T3 RNA polymerase) from appropriately cleaved pSH2. The RNA transcripts contain 5* exon, part of the intron containing domain 1 - 3 plus varying amounts of domain 4 and constitute the upstream "half molecules" in the trans splicing experiments. Next, the Cla I to Eco RI fragment of pSH2 (Figure 10) was subcloned into pBSM13+ to yield plasmid pSH4. It was linearized at Eco RI and transcribed with T3 RNA polymerase to yield the 650 nt 3* "half molecule" containing the 63 terminal 274 nt of domain 4, all of domains 5 and 6 plus the 3* exon. Because the 3* half molecule is only slightly shorter than spliced exons, the trans-splicing experiments shown employed roughly equimolar mixtures of 5* and 3* half molecules where the former was randomly labeled with 32p UTP and the latter, unlabeled. Initial studies showed that all four half molecules are unreactive when incubated singly under both NH«C1 and KC1 conditions (not shown). In the first trans-splicing experiment, the 5* half molecule ends at the Cla I site in domain 4 and the 3’ half molecule provides all intron and exon sequences missing from the 5* one. Control samples containing the full length precursor RNA were incubated in NH4 CI (Figure 39, lane 1) and in KC1 (lane 2) while in lane 3 the partial precursors were mixed but not incubated. The 5* half molecule terminated at the Clal site was incubated with roughly equimolar quantities of the 3* half molecule in 1 M NH« Cl (lane 4) and in 1 M KC1 (lane 5) . It is clear that the two "half" molecules interact to yield product bands. As noted alongside lane 1, products co-migrating with the 5* exon and spliced exons are prominent in both reacted samples. Both reactions contain a product that is the correct size for the excised intron from the 5’ half molecule. Also, the reaction in the presence of KC1 contains additional products that comigrate with products of the control reaction (compare lanes 1 and 5). 64 A second set of trans-splicing reactions using the 5* half molecule terminated at the Acc I site, that lacks 1107 nts of domain, 4 still yields the same array of products (Figure 40, lanes 3-5) as the wild type precursor RNA (lanes 1 and 2). The product identified as excised intron RNA (lanes 1 and 2) is absent and replaced by a new band about 1100 nt smaller (lanes 3 and 4). When the 5* half molecule terminating at the intron Sea I site, deleted for 1584 nt of domain 4, was used similar products were observed (lanes 6-8). Although all the exon-containing products of these trans-reactions were identified only by co-migration with well characterized products of the control cis-reactions, it is highly likely that this group IIA intron, like aI5g, trans-splices and that the majority of domain 4 of all is not essential for in vitro trans- reactions. III.G.2. The conserved 5'boundary sequence is needed for trans-splicing Direct proof that a group II intron recognizes its own 5' exon was provided by the trans-splicing experiments of Jacquier and Rosbash (1986), in which co-incubation of the 5' exon and IVS-3’E transcripts (splicing intermediates of the first step) resulted in efficient production of spliced exons and free, linear intron. To test whether all would carry out a similar reaction, I wanted to do the analogous experiment using all 5*E and IVS-3'E. It was difficult to 65 obtain large amounts of clean all IVS-3’E free of contaminating precursor RNA or IVS-BL because of the proximity of the bands, so that approach yielded equivocal results. An alternative approach to this goal was then taken. In order to be able to synthesize IVS-3’E in vitro, plasmid pSH3 was constructed by subcloning the Hha I - Hha I fragment from the original plasmid pSH2 into pBSM13+ cleaved by Acc I (Figure 10). Plasmids in both orientations (behind the T3 or T7 promoter) were identified by restriction mapping. Construction a ? these plasmids resulted in the replacement of the 5* exon by 27 nt of polylinker sequence and deletion of a single nucleotide from the highly conserved 5' intron sequence GUGCG (guanosine residue was deleted). Wild type TTAATG/GUGCGC T3 CTGCAG/GU-1CGC / denotes the 5*splice junction. All sequences downstream of CGC are wildtype. RNA transcribed from plasmid pSH3 (behind T3 polymerase) was inactive when incubated by itself in the presence of NH«.C1 (Figure 41, lane 6). When the IVS-3*E RNA was incubated with an excess (30 fold) of 5'E (lane 5), the RNA remained inactive and the reaction did not result in productive trans-splicing. Since the only difference between this and Jaquier’s trans-splicing experiment is the 66 deletion of a single nt from the IVS-3’E, it suggests that the highly conserved GUGCG plays a role in trans-splicing. While the deletion of a single nt from the GUGCG sequence has a dramatic effect on trans-splicing, it would be interesting to examine if it would affect splicing in cis. This issue, however, has not been addressed experimentally in this thesis. III.H. Summary I have clearly demonstrated that much of the ORF (intron domain 4) of all is dispensible. This lends support to the earlier observation (Jarrell et al., 1988b) that domain 4 is not essential for the in vitro reactivity of aI5g. The shortened all intron (plasmid pSH AC) is more amenable to in vitro characterization than the full-length intron. Branch point analysis were done using the deleted all intron and the branch residue mapped to the last 9 nt of the intron. I have shown that all, like aI5g, trans splices in vitro. In addition, I have demonstrated that all carries out a unique post-splicing reaction in KC1 and that unlike group IIB introns (aI5g and bll) it does not carry out SER. 67 111.1. Studies of aI5g self-splicing 111.1.1. Introduction Using 3’ intron truncations Jarrell (1987) showed that precursor RNAs lacking portions of the 3* end of the intron (part or all of domain 6) catalyze step 1 of splicing — cleavage at the 5* splice junction, while precursors lacking domain 5 cannot catalyze the reaction. A bimolecular model for this reaction confirms that domain 6 is dispensible while domain 5 is required for the first step in splicing (Jarrell et al„, 1988b). They demonstrated that a short, 42 nt long, RNA species containing only domain 5 sequences, when supplied as a trans-acting RNA, initiates the first step in splicing --- cleavage at the 5* splice site. This set the stage for a comprehensive analysis of the role of domain 5 RNA in the first step of splicing. Having established that all self-splices in vitro, I decided to study domain 5 function (and several closely related issues) using aI5g because it is the most intensely studied self-splicing group II intron and its size (900 bp) makes it more amenable to in vitro dissection and characterization than all (2448 bp). Three questions need to be addressed: 1) which portions of domain 5 are essential? 2) where does domain 5 interact? 3) how does domain 5 activate the 5* splice site? 68 III.1.2. Doiain 5 is required, in cis, for the second step of splicing As confirmation that domain 5 is required for the first step in splicing, I cleanly deleted domain 5 in cis from the plasmid containing the full-length precursor by site-directed mutagenesis to create plasmid pSH £ 5 (see Materials and Methods). In this construct, the 2 nt at the base of the helix were retained so that the spacing between domains 4 and 6 remains unaltered. Clones deleted for domain 5 were screened for loss of the domain 5 Rsa I site and confirmed by dideoxy sequencing across the deletion (Figure 42). To study the effect of the deletion, RNA transcripts lacking domain 5 (from plasmid pSI!15) were analysed under the standard splicing conditions (Figure 43A, lane 4), 100 mM MgCla (lane 5), 0.5 M (NHOaSO* and 0.5 M KC1 (lanes 6 and 7 respectively). Unreacted RNA in lane 3 and samples with full-length precursor reacted in (NH«)2 SO« and KC1 (lane 1 and 2 respectively) are shown as controls. As expected, RNA lacking domain 5 was inactive under all conditions examined confirming that domain 5 is essential for initiating the first step in splicing. If domain 5 alone is sufficient to initiate splicing it is reasonable to assume that the defect caused by a cis deletion of domain 5 could be suppressed by supplying 69 domain 5 in trans. This was tested directly by incubating essentially pure unlabeled domain 5 RNA (1VS 5 RNA), transcribed from plasmid pJDI5*-75 (see Jarrell et al., 1988b), with uniformly labeled precursor RNA lacking domain 5 (Figure 43B). Precursor RNA lacking domain 5 was either not reacted (lane 6) or reacted in KC1 (lane 7). Only samples of RNA incubated with an excess (30 fold) of unlabeled domain 5 RNA (lane 8) were cleaved at the 5 * splice site to yield free 5*E and IVS-3*E. There was, however, no product the size of spliced exons nor was there any lariat form of IVS-3’E. In lanes 3-5, E5(IVS 1-3) RNA (5’ half molecule from which sequences 3’ of domain 3 are deleted) was used as a positive control (Jarrell et al., 1988b). The absence of spliced exons among the reaction products was surprising. The reaction intermediates, 5’E and IVS-3'E, appear for some reason unable to carry out the second step of splicing — cleavage at the 3* splice-site followed by exon ligation. Clearly, an intron branch is not an essential prerequiste for the second step under these and related conditions (Jacquier et al., 1986; Jarrell et al., 1988a). Yet the removal of domain 5 blocks that step completely. This suggests that domain 5 may also play a role in the second step of splicing. It is possible that the absence of domain 5 from the IVS-3'E RNA impairs its ability to fold into a productive confirmation that 70 would make the 3* splice site accessible to cleavage. C. Intact domain 3 is required for S' cleavage It is interesting to note that interruption of the precursor RNA in domain 3 does not support trans-splicing (K. Jarrell, Unpublished). I extended this observation by demonstrating that domain 5, when supplied in trans, is unable to release free 5* exon from a 5’ half molecule that is interrupted in domain 3. RNA transcribed from plasmid pJD20-Xba I (R. Dietrich, Unpublished) linearized at Xba I was either not reacted (Figure 44, lane 5), reacted in KC1 (lane 6) or reacted in KC1 with an excess of IVS 5 RNA (lane 7) . As shown in lane 7, the IVS 5 RNA ,-was unable to cleave at the 5* splice junction. This indicates that domain 5 requires an intact domain 3 to carry out the first step in splicing. As a control, TVS 5 RNA is shown (lane 4) to cleave at the splice junction of E5(IVS 1-3) RNA to yield 5* exon plus excised IVS 1-3. In addition, in vivo point mutations in domain 3 have been reported for both aI5g and bll, providing further evidence for the, functional importance of this domain. 111.1.3. Mutational analysis of the conserved 5* end of the intron 111.1.3.a. The first 7 nt of the intron are essential for branch formation The data presented above lend support to the previous conclusion that domain 5 is necessary for the first step in splicing. They are the strongest evidence that it plays a role in the second step as well. The obvious question that needs to be addressed is — where does domain 5 interact to activate the 5* splice site for cleavage? Since all group II introns possess a short conserved sequence at their 5’ ends, the specificity of this attack at the 5* splice-site could be provided by sequences around the exon intron junction. Jarrell (1987) proposed a model in which the 5* half of the bottom helix of domain 5 base pairs with the first 7 nt of the intron (Figure 45) and by so doing in some way activates the 5* splice-site for cleavage. He considered the possibility that part of domain 5 may bind Mg2* and that the proposed pairing delivers the needed Mg2* to the reaction site. A direct test of that model was begun by R. Dietrich (Unpublished) who used site-directed mutagenesis to change the first nucleotide of the intron from a G to each of the other three nucleotides, A, C or U. Incidentally, as seen below, those experiments constitute the first analysis of 72 the role of the conserved sequence at the 5* ends of group II introns. Surprisingly, those first intron base (FIB) mutations did not affect the efficiency of 5* exon release or splicing. The efficiency of branching was reduced somewhat in (NHt^SO* and more dramatically in KC1 (essentially all of the excised intron being linear compared with about 2/3 using wild type precursor). Based on this result it is likely that domain 5 does not interact at the boundary, or if it interacts, the interaction is more important for branching than for 5’ exon release. This is in sharp contra * to nuclear pre-mRNA introns which fail to splice correctly when the first intron base is mutated (Jacquier et ale, 1985). The fact that the FIB mutants yield spliced exons and more importantly free exons from SER that are normal in size suggests that the 5* part of each mutant branch is certainly the expected new base. Assuming that these mutants still form branches using the adenosine residue (branch site), the IVS-LAR RNAs obtained with them should contain novel branch points (A 2',5* A, A 2*,5* C and A 2’,5’ U) that differ from the conventional A 2’,5* G branch. I examined the susceptibility of these mutant IVS- LARs to the Hela S100 debranching enzyme (Figure 46). The FIB IVS-LABs were purified and samples were either not incubated (lanes 2, 5, 8 and 11) or incubated in the presence of the debranching extract (lanes 3, 6, 9 and 12). 73 As a control IVS-LAR from the wild type precursor RNA is shown {lane 3) to debranch when treated with the debranching actvity, yielding a product that comigrates with the IVS-BL. The FIB IVS-LARs (lanes 6, 9 and 12) on the other hand, remained unaffected by treatment with the debranching extract suggesting that the extract may be specific for a A 2*,5* G branch. Ruskin et al., (1985) showed that both the A 2* ,5* G and the A 2 *, 5, A lariata are completely hydrolyzed when tested in the crude extract and concluded that the debranching extract lacks a stringent requirement for the nucleotide composition of the 2',5* phosp adiester bGnd. They, however, pointed out that additional substrates need to be analyzed with purified or partially purified debranching enzyme to confirm their conclusions. My result with the A 2’,5* A lariat is in conflict with the published result or the FIB-IVS LARs do not involve the domain 6 adenosine residue. One possible explanation for this difference in substrate specificity could be that the debranching enzyme used in my experiments is highly purified (a gift from Mary Edmonds, at the University of Pittsburgh) while the activity used in the original study was from a crude extract. The finding that changing the first nucleotide of the intron has little effect on the efficiency of 5* exon release was rather unexpected. It, however, does not rule 74 out the proposed model that domain 5 interacts with the first 7 nt of the intron. It could be possible that this interaction is sufficiently strong and that more than one nucleotide needs to be changed in order to block 5* exon release. The finding that the highly conserved 5* G residue is not essential for RNA catalysis, made it necessary to learn whether the rest of the 5* consensus sequence is important for the in vitro reactions. I tested this point by changing the next 6 nt AGCGGU to CUGCAG (a Pst I site) by site directed mutagenesis. Formation of the Pst I site (plasmid pSH5'I~PstI) was checked by restriction enzyme analysis and confirimed by dideoxy sequencing (Figure 47). If the first few nucleotides of the intron are critical for 5* cleavage, RNA from pSH5’I-PstI should be inactive under all splicing conditions. The RNA was found to be inactive when incubated in 100 mM Mg2* (Figure 48, lane 2) and in 0.5 M (NEUJzSCU (lane 3). But to our surprise, the mutant RNA was partially reactive in the presence of 0.5 M KC1 (lane 4) yielding spliced exons and IVS-LIN RNA without lariat (branch) formation. The wild type precursor RNA, on the other hand, yields mostly free 5*E and 3'E in KC1 and very little spliced exons becai.i>j of the ability of the intron to carry out SER. It was, therefore, intriguing to find that the mutant RNA yields spliced exons in the presence of KC1. A reasonable way to 75 account for the absence of free exons among the reaction products is to assume that the mutant intron is incapable of carrying out the SER reaction. The identity of the spliced exons was checked by its susceptibility to cleavage by wild type IVS-BL to yield free exons (see next section). III.I.3.b. 5’ end of the intron plays a role in SER To test whether the mutant RNA (pSH5’I-PstI) is blocked in SER, both wild type and mutant IVS-LIN and 5*E- 3*E RNAs were isolated from preparative polyacrylamide - urea gels. The 5’E-3’E RNAs were mixed with the IVS-LIN RNAs and samples were either unreacted (Figure 49, lanes 1, 3, 5 and 7) or reacted in the presence of KC1 (lanes 2, 4, 6 and L As shown, the wildtype enzyme (IVS-BL) carries out the SER reaction efficiently on the spliced exons from both the wildtype and the mutant precursor (lanes 2 and 4). The mutant enzyme (IVS-LIN) is, however incapable of carrying out SER on spliced exons from the wild type or the mutant precursor (lanes 6 and 8). This confirms my conclusion that the first few nucleotides of the intron play an important role in SER. We have, therefore, fortuitously, hit upon a specific sequence within the intron which, when mutated blocks the ability of the intron to carry out SER. 76 III.I.4. Donain 6 is not required for splicing in vitro To investigate the role of lariat formation further, I deleted intron domain 6 (branch helix) by site-directed mutagenesis. In this construct (plasmid pSHA6) the 2 nt at the base of the helix were retained. Possible candidates deleted for domain 6 were screened for loss of the domain 6 Acc I site and confirmed by dideoxy sequencing (Figure 50). In vitro analysis of precursor RNA lacking domain 6 (from plasmid pSH 6) is shown in Figure 51. The RNA was inactive under the standard splicing conditions (lane 4) and in 100 mM Mg2 + (lane 5). Interestingly, the RNA splices efficiently without lariat formation in 0.5 M (NHOsSP* (lane 6) and in 0.45M KC1 (lane 7) suggesting that the highly conserved branch helix (or domain 6) is not required for either the first or the second step of splicing. Clearly, the proximity of domain 6 to 3'E is not the mechanism by which 3’E is attached. Samples containing unreacted RNA (lane 3) and the wild type precursor RNA reacted in (NHf^SO* and KC1 (lane 1 and 2 repectively) are shown as controls. 77 III.J. Molecular dissection of donain 5 III.J.1. Introduction Intron domain 5 is the most highly conserved structure of group II introns. In most cases the sequence is 34 nt long and is capable of forming a stem - loop structure with 14 basepairsi a 4 base loop and a 2 base bulge (Figure 52). The unpaired bases and the lower half of the helix, through the bulge are very highly conserved. The least highly conserved region is the top helix adjacent to the 4 base loop. Such variability in primary sequence suggests that it is the secondary or higher ordered structure rather than the particular nucleotide sequence that is important in this part of domain 5. Alternatively, the variable elements might relate to additional, intron-specific functions of domain 5. III.J.2. Heterologous experiments A comparison of the sequence of domain 5 of s,I5g with that of all (Figure 53) shows that the two structures differ at 9 positions, 7 of which are in the top half of the helix. Since the two sequences are still so similar, inspite of the differences, I reasoned that domain 5 of aI5g might work in a heterologous experiment with all RNA that lacks its own domain 5. Taking advantage of a unique Cla I site within domain 4 of all (Figure 10), uniformly labeled run-off transcripts were synthesized using T3 RNA 78 polymerase. The Cla I run-off RNA contains 5* exon, intron domains 1 - 3 and part of domain 4. Samples of this RNA were either not reacted (Figure 54, lane 1), reacted in KC1 (lane 2) or incubated with an excess (30 fold) of unlabeled al5g domain 5 RNA (lane 3). All KC1 reaction products were run alongside as a control (lane 4). As shown in lane 3, aI5g domain 5 RNA when incubated with a deleted form of all lacking its own domain 5, releases a product that co- migrates with free 5* exon. This indicates that domain 5 of aI5g stimulates, in trans, a cleavage at or very near the 5’ splice site of the all RNA. Since domain 5 of these two introns differs mainly in the top half of the helix, the data suggest that the specific sequence of the top half of the helix of domain 5 is not important for 5’ exon release. This result also extends the conclusion that domain 5 is needed for step 1, by a group IIA intron. III.J.3. Mutations of the highly conserved unpaired regions of domain 5 III.J.3.a. The 4 base pair loop It is reasonable to assume that domain 5 structural elements that are conserved among group II introns will prove to be functionally important. Mutational analyses were carried out to examine the significance of these conserved elements. During the course of site-directed modifications of domain 5, the highly conserved 4 nt loop GAAA was changed to GAUC and the closing base pair U:G to 79 G:C, creating a Bam I site within the loop sequence (plasmid pSH-Bam I). This was confirmed by restriction analysis and dideoxy sequencing (Figure 55A). In vitro analysis shows that the RNA from plasmid pSH-Bam I is inactive in the standard splicing buffer (Figure 56, lane 9) and in 100 mM MgCla (lane 10) but was, surprisingly, wild type in the presence of 0.5 M (NHi^SO* (lane 11) and 0.5 M KC1 (lane 12). Taking advantage of the Bam HI site, I cleaved plasmid pSH-Bam HI with Bam HI, filled in using the Klenow fragment of Pol I and dNTPs and ligated the ends using DNA ligase (plasmid pSH-Bam 4). Clones were screened for the loss of domain 5 Bam HI restriction site and confirmed by dideoxy sequencing (Figure 55B). RNA transcribed from plasmid pSH- Bam 4 has a 4 base TCGA loop in domain 5 and the top helix is extended by 2 base-pairs. In vitro analysis of the RNA shows that the RNA is inactive in the standard splicing conditions (Figure 56, lane 4) and in 100 mM Mga* (lane 5) but is weakly reactive in 0.5 M (NH4 )2 SO* (lane 6 ). Inspite of the drastic alteration in the loop sequence and the top helix of domain 5, the °:;^ant RNA appears almost wild type in 0.5 M KC1 (lane 7). 80 III.J.3.b. RNA truncated at the Rsa I site in domain 5 is inactive Jarrell (1987) showed that the rate of 5*E release from RNA truncated at the Rsa I site (plasmid pJDI3*-839) in domain 5 is about 5-fold slower than wild type. ‘This suggested that the last 7 nt of domain 5 are not essential for the first step in splicing. Having inserted a Bam HI site (plasmid pSH-Bam HI) in the 4 base loop of domain 5, I wanted to ask if all of the 3* half of domain 5 is dispensible. Run-off transcripts from plasmid pJD2 linearized at Rsa I and plasmid pSH-Bam I linearized at Bam HI were used in the following experiment (Figure 57). Surprisingly, the Bam HI run-off transcript was inactive both in (NH«)2 S0 « (Figure 58, lane 11) and in KC1 (lane 12) suggesting that sequences in the 3* half of domain 5 are required for the first step in splicing. RNA from the Hpa II truncation (plasmid pJDI3*-851) was used as a positive control (Jarrell, 1987). While the Hpa II truncation (lacks sequences 3’ of the Hpa II site in domain 6 ) releases 5’E as efficiently as the full-length precursor RNA (lanes 1-3), Rsa I run-off transcript was inactive both in ( N H O 2 SO4 (lane 8 ) and in KC1 (lane 9). This discrepancy concerning the Rsa I runoff transcript may be due to the fact that the RNA used by Jarrell (1987) was not a Rsa I run-off transcript but RNA transcribed from plasmid 81 pJDI3'-839 linearized at Hind III. This RNA has additional nucleotides (polylinker sequence) 3* of the Rsa I site which may aid in partially restoring the bottom helix, accounting for the reactivity of the RNA in vitro. At a recent meeting O.Uhlenbeck (University of Colarado) demonstrated that the reactivity of some RNA molecules can be enhanced by re-annealing the RNA in the splicing buffer (RNA is heated at 95°C for 3 minutes and then immediately lowered to 20°C for 5 minutes) prior to the in vitro reactions. In order to resolve the inactivity of the Rsa I run-off transcript, I reannealed the RNA before reacting it but failed to detect any activity (not shown). Since the Rsa I runoff transcript is inactive, it suggests that an intact domain 5 is required for function. This interpretation is further supported by the fact that a linker insertion at the Rsa I site completely inactivates the intron in vitro (Dietrich, Unpublished). III.J.3.c. The CG bulge The two-base CG bulge in domain 5 is highly conserved among group II introns. Bulged nucleotides tend to destabilize helix structures (Tuerk et al., 1988) and are considered to be potential sites for interaction with proteins or nucleic acids. To investigate the role played by the CG bulge, I deleted it by site-directed mutagenesis. The deletion (plasmid pSHACG) was initially screened for loss of the domain 5 Rsa I site and later confirmed by 82 dideoxy sequencing (Figure 59A). Uniformly labeled pSHACG RNA was either not incubated (Figure 60A, lane 1), incubated in the standard splicing buffer (lane 2 ), in 1 0 0 mM MgCl* (lane 3), in 0.5 M (NH« )2 SO4 or incubated in 0.5 M KC1 (lane 5). Interestingly, RNA lacking the CG bulge was inactive under all conditions (lanes 2-5). It is possible that the deletion stabilizes the structure of the helix significantly in a manner that affects the flexibility of domain 5, preventing it from carrying out its normal function. In order to determine whether it is the sequence of the bulge or the presence of the bulge per se that is important, I changed the CG to a GC bulge by site-directed mutagenesis (plasmid pSH-GC). Clones were screened for the loss of the domain 5 Rsa I site and confirmed by dideoxy sequencing (Figure 59B). In vitro analysis of RNA transcribed from plasmid pSH-GC shows that the mutant RNA is unreactive under the standard splicing condition (Figure 60B, lane 2) and in 100 mM Mg2* (lane 3) but is perfectly wildtype in 0.5 M (NH«)2 SO« (lane 4) and in 0.5 M KC1 (lane 5). This suggests that it is the presence of the bulge rather than its sequence that is crucial for function of domain 5. This observation was further confirmed by altering the CG bulge to a UC bulge (plasmid pSH-UC). Clones were screened for loss of the domain 5 Rsa I restriction site 83 and confirmed by dideoxy sequencing (Figure 59C). RNA transcripts from plasmid pSH-UC, similar to RNA precursors with a GC bulge, is reactive under the high salt conditions (Figure 60C, lanes 4 and 5) but are inactive under the standard (lane 2) and the high Mg3* (lane 3) splicing conditions. III.J.4. Bottom helix As pointed out earlier the sequence of the 5’ half of the bottom helix can potentially basepair with the first six nucleotides of the 5* end of the intron. I have already established that the highly conserved sequence at the 5* end of the intron is not essential for 5* cleavage or splicing in KC1. Since the bottom half of the helix is highly conserved among group II introns, I altered the sequence in the 5* half of the bottom helix by site- directed mutagenesis. The sequence GCCGUA was changed to CUGCAG, creating a Pst I site (Figure 61). The mutation was confirmed by restriction mapping and sequencing (Figure 62A). Uniformly labeled RNA from plasmid pSH5'PstI, was either not reacted (Figure 63, lane 3), reacted in the standard splicing buffer (lane 4), in 100 mM MgC12 (lane 5), in 0.5 M (NH4)2S04 (lane 6 ) and in 0.5 M KC1 (lane 7). As shown, the mutant RNA was inactive under all splicing conditions (lanes 4 - 7). Similar to the pSH5’PstI (domain 5) mutation I constructed plasmid pSH3*PstI by site-directed mutagenesis, 84 in which the sequence UACGGU in the 3* half of the bottom helix was changed to CUGCAG (a Pst I site ). The mutation / was screened by restriction analysis and confirmed by dideoxy sequencing (Figure 62B). Similar to the 5' Pst I mutation, the 3* mutant RNA was inactive under all conditions (Figure 63, lanes 8-12). This suggests that the sequence of the bottom helix may be important for function. Alternatively, it could be possible that a mutation of this magnitude (Figure 61) may completely disrupt the pairing of the bottom helix thus destabilizing the secondary structure of domain 5 critical for function. It is also possible that this change reconfigures the intron more globally, blocking function. To test this, I examined whether the 42 nt long, domain 5 RNA could suppress, in trans, the mutant phenotype of pSH5’PstI RNA and pSH3’-PstI RNA. Samples of internally labeled pSH5*PstI RNA and pSH3'-PstI RNA were either not,, reacted (Figure 64, lane 6 and 9), reacted in 0.5 M KCl (lanes 7 and 10) or incubated with an excess of unlabeled domain 5 RNA (lanes 8 and 11). As expected, domain 5 RNA restored the first step in splicing (lanes 8 and 11). The free 5* exon released from the first step was, however, unable to cleave the 3’ splice-site of the mutant IVS-3’E . This indicates that the presence of a defective domain 5 ii cis precludes 3* cleavage and exon ligation, consistent with our earlier observation that domain 5 may also be 35 required for the second step in splicing. III.J.5. 7 nt in the 5* half of domain 5 can fora a perfect match with 7nt in domain 1 Based on my observations, the original model for the possible interaction of domain 5 with the 5* end of the intron does not hold strong. A search for potential base pairing between domain 5 and the upstream molecule showed that a 7 nt sequence in domain 1 can form a perfect match with 7 nt in domain 5 (Figure 65). To test this interaction I constructed two point mutations in domain 5 that would disrupt the proposed base pairing (Figure 6 6 ). First, the single U residue in the 5 ’ half of the bottom helix was changed to a C. This creates an Sph I site that was used for screening the mutants. The mutation was confirmed by dideoxy sequencing (Figure 67B). Interestingly, in the region S’AGCCGC 3', CC always appears as a single intense band on the sequencing gel instead of a doublet. If, indeed, there was a fortuitous deletion of a single C, it would create a Hha I site (GCGC). By restriction analysis I confirmed that there is no Hha I site in domain 5, establishing that the pSH-Sph I RNA contains only a single U to C change. Uniformly labeled pSH-Sphl RNA was analysed in vitro (Figure 6 8 , lanes 8-12). Contrary to our expectation, the mutant RNA is reactive in 0.5 M KCl (lane 8 ), in 0.5 M (NH«)2 S04 (lane 9) and in 100 mM Mg2+. The RNA is, however, inactive under the standard 86 splicing conditions (lane 1 1 ). Second, a U residue in the top helix (5* AUGCG 3’) was changed to a C (5’ ACGCG 3*), creating a Tha I site that was confirmed by DMA sequencing (Figure 67A). RNA transcribed from plasmid pSH-Tha I is weakly reactive in 0.5 M KC1 (Figure 6 8 , lane 3) and in 0.5 M (NH«)2 S0 « (lane 4) and inactive both in 100 mM Mg2* and in the standard splicing buffer (lane 5 & 6 respectively). Wild type RNA reacted in (NH«)2 S0 « (lane 1) and KC1 (lane 2) was used as controls. The lack of a strong mutant phenotype in the case of the Sph I mutant could be because the C/A juxtaposition does not severely disrupt the proposed pairing. In vivo there are RNA helices that contain G/G, U/U, A/C and G/A "pairs" (James et al., 1988). Such irregularities need not render helices unstable and could be the explanation for the almost wild type phenotype of the Sph I mutant. Interestingly, the Tha I mutant is only weakly reactive under the high salt conditions suggesting that the U to a C change in domain 5 is critical for the in vitro reaction. As a first step towards establishing the importance of the seven nucleotides (5 * GCAUACG 3 * ) in domain 1, I altered the sequence by site directed mutagenesis to 5’ AUAUACC 3’ (plasmid pSH-Doml)and confirmed it by dideoxy sequencing (Figure 69). Since the underlined nucleotides (GC and G) are unpaired, the mutation (pSH-Doml) should not 87 alter the secondary structure of the intron. One would expect, such a mutation to be of little consequence unless the nucleotides are critical for the self-splicing reaction or for the proposed interaction with domain 5. RNA transcribed from plasmid pSH-Doml was inactive in the low salt and the high Mg*+ splicing buffers (Figure 70, lanes 4 and 5 ) but the RNA was almost wild type in 0.5 M (NH«)2 SO« (lane 6 ) and in 0.5 M EC1 (lane 7). The lack of a definitive phenotype suggests that additional mutations and compensating changes need to be constructed in domains 1 and 5. These experiments are clearly beyond the scope of this dissertation. CHAPTER IV DISCUSSION IV.A. All self-splices in vitro In section III.C., I have shown that the first intron of the cox I gene of yeast ratDNA, a maturase encoding group II intron (Carignani et al., 1983) self-splices efficiently in vitro to yield accurately spliced exons and excised intron lariat. This is the first maturase encoding intron of either group I or II shown to self-splice in vitro. All is also the first example of a member of the large subgroup IIA to be shown to self-splice. I have clearly demonstrated that the in vivo dependence of the all intron on its own maturase does not preclude self-splicing in vitro. The question then arises: what is the role of the maturase in vivo? Several lines of evidence strongly suggest that "maturases" are required for mtRNA splicing in vivo (reviewed by Kotylak et al., 1135; Banroques et al., 1986). Since intron-encoded maturases are for most part intron- specific, it has been considered unlikely that they are general splicing enzymes (Lazowska et al., 1980) and are 88 89 thought to be involved in stabilizing the secondary structure of the RNA (Davies et al., 1982). The exact function of the maturases, however, remains a matter of speculation. The observation that all self-splices in vitro does not, however, rule out the involvement of its maturase in vivo. The need for unusually high concentrations of salt (1M) for maximal activity in vitro is probably due in part to a requirement for electrostatic screening. Since the RNA is negatively charged it would otherwise tend to repel itself thus destabilizing the secondary and tertiary interactions crucial for the self-splicing reaction. The salts thus appear to provide structural stability, leading to the inference that this may be the role of the maturase in vivo. Since members of both group IIA (all) and I IB (aI5g and bll) self-splice in vitro, it is possible that the self-splicing reaction represents the basic splicing mechanism in vivo and that additional trans-acting factors (presumably proteins) facilitate these reactions by inducing or stabilizing the productive folding of the RNA or by accelerating the rate of the reaction in vivo. 90 IV.A.l. All self-splices under conditions different from those of al5g and bll From studies of reaction conditions, the optimum in vitro splicing condition for all was established as 1 0 0 mM MgCl2 , 1 M NH«C1, 40 mM Tris-chloride (pH 7.5) and 40°C. Clearly all, a group IIA intron, self-splices under conditions somewhat different from those of the self splicing group IIB introns, aI5g and bll (Peebles et al., 1986; Schmelzer et al., 1986; van der Veen et al. , 1986 ; Jarrell et al., 1987). Specifically, all has a temperature optimum of 40°C, 5°C lower than the sharp temperature optimum of 45°C for aI5g and bll. In addition, all has a higher threshold for magnesium. While aI5g, in the presence of 2mM spermidine, is reactive throughout the range of 10 - 100 mM Mg2*, all is inactive even at 100 mM Mg2* . Interestingly all, unlike aI5g and bll, has an absolute requirement for monovalent cations. The optimum all salt concentration is 2-fold higher (1M) than for aI5g (Jarrell et al., 1988a). This difference in requirements of Mg2* and monovalent cations in the self-splicing reaction of all and other group IIB introns may reflect a requirement for greater electrostatic screening since the all intron is almost three times larger (2.4 Kb) than aI5g or bll (0.9 Kb). In fact increasing the concentration of Mg2* has been shown to stabilize secondary structure of the Tetrahymena rRNA intron (Inoue et al., 1985). IV.A.2. All reaction pathways Group II introns self-splice to yield spliced exons and excised intron lariat. These two products correspond to the final products of nuclear pre-mRNA splicing. In the initial study of aI5g self-splicing no intermediates were detected, indicating that the reaction is highly coupled. In contrast, all splicing intermediates, free 5’E and IVS- 3’E RNA are prominent among the reaction products. Thus, the all self-splicing pathway appears to involve a bipartite splicing intermediate similar to nuclear introns. It is important to note that while IVS-3*E is stable among the reaction products, IVS-3’E-LAR is not detectable. It is possible that in step 2 (3* cleavage and exon ligation) the lariat form is more reactive than the linear one (Jarrell et al., 1988a). 92 IV.A.3* All undergoes a post-splicing reaction in KC.1 I have shown (section III.D.) that the addition of EC1 to the all splicing buffer dramatically alters the pattern of products relative to reactions containing NH«C1. In addition to the set of products common to the two conditions (5*E, S'E-S'E, IVS-BIi) , the precursor RNA in KC1 yields novel intron lariats and 51intron fragments. The overall all splicing mechanism in KC1 is similar to that of aI5g (cleavage at the 5* boundary occurs by hydrolysis rather than by transesterification). Since the attacking nucleophile in the first step is a water molecule and not the 2 ’-OH group of the branch residue, excised intron is released as a linear molecule rather than a lariat. The linear intron, when incubated in KC1, is capable of cleaving itself at either of two sites near its 5* end yielding 5' intron fragments and IVS-LARs smaller than the full-length IVS-LAR. This is the first example of a specific intramolecular transesterification reaction carried out by an excised group II intron and may be analogous to some post-splicing reactions of group I introns (Zaug et al., 1983). Post-splicing reactions of aI5g IVS-LIN exist but are very different (Jarrell, 1987). 93 IV.A.4. All does not carry out spliced exon reopening The SER reaction accounts for the appearance of separate 5* and 3’E RNAs among the KC1 reaction products of aI5g and bll (Jarrell et al., 1988a, R. Dietrich, Unpublished). In the case of all, free 3 ’E is notable by its absence while free 5*E and IVS-3’E present in reacted samples are probably reaction intermediates. I have shown that all intron RNA does not reopen its spliced exon3 . It, therefore, follows that free 3'E is absent from the splicing products. I have demonstrated that the ability to reopen spliced exons is sequence specific. The fact that both aI5g and bll (group IIB introns) carry out SER but all (group IIA intron) does not, suggests that it may be a subgroup specific feature. IV.A.5. Much of the intron open reading frame can be deleted As was found for aI5g (Jarrell et al., 1988b), interruption of the all precursor RNA in domain 4 allows trans-splicing in vitro. Deletions of large portions of the all ORF (most of domain 4) was with little effect on trans-splicing. Incidentally, this is the first time that a group IIA intron has been shown to trans-splice. In addition, a cis deletion of over half (60%) of intron domain 4 did not alter the autocatalytic ability of the intron. These results are consistent with the observation 94 that domain 4 of aI5g can be deleted without any effect on the in vitro reaction (Jarrell et al., 1988b). Among group 11 introns, domain 4 appears to be a non- essential region; nonetheless, it is conserved. It is likely that, in the course of evolution, intron domain 4 is a region into which sequences can be inserted or deleted without affecting the splicing reaction. Now that methods for transforming yeast mtDNA have been developed (Johnston et al., 1988) it will be possible to test the role of domain 4 under in vivo conditions using the same intron derivatives. IV.B. 5* end of the intron is not required for 5 ’ exon release but plays a role in branch formation Having demonstrated several features of the all self- splicing reaction, I decided to investigate the function of intron domains. Prompted by the recent finding (Jarrell et al., 1988b) that domain 5 can promote 5* exon release when supplied a 3 a 42 nt trans acting RNA, I decided to study the role of domain 5 and other related issues using a!5g. In fact, structural studies of all (even with the shortened intron form) would be quite complex because of its large size. Jarrell (1987) proposed a model in which domain 5 can interact with the first 7 nt of the intron. I have shown that mutation of the conserved sequence at the 5’ end of the intron (plasmid pSHI5*-Pst I) inactivates the intron 95 under all conditions except KC1 (Figure 48). In KCl, the RNA is somewhat reactive yielding spliced exons without lariat formation. The ability of the mutant RNA to carry out splicing in KCl suggests that the nucleotides at the 5' end of the intron are not absolutely essential for splice- site selection, ruling out the model proposed by Jarrell (1987). The fact that pSH5*I-PstI RNA yields spliced exons in KCl without lariat formation together with the data on the first intron base mutants (which reduce the efficiency of branch formation without affecting the efficiency of splicing) suggests that the nucleotides at the 5* end of the intron play a significant role in branch formation but are not absolutely essential for splice site selection. Our interpretation that the 5* end of the intron is essential for lariat formation.is supported by the recent demonstration (M. Rosbash, personal communication) that the nucleotides at the 5 * end of the intron are involved in suppressing the cleavage at the 5’ spiice-site by hydrolysis in favor of branch-cleavage. 96 IV.C*. 5* end of the intron plays a role in SER The observation that mutation at the 5* and of the intron (plasmid pSH 5’1-Pst I) causes an accumulation of spliced exons in KCl led to the exciting possibility that the mutant RNA is incapable of carrying out SER in KCl. This was indeed true (Figure 49). I have, therefore, defined a specific region (6 nucleotides) of the intron that is essential for SER. Previously, Jarrell et al., (1988b) have proposed that SER is related to the second step of splicing (abortive reversal of step 2 by hydrolysis rather than by transesterification). The present result is a first indication that either their proposal is incorrect, or else the 5* conserved sequence is involved in both splicing steps. This reaction is now being investigated in detail by Craig Peebles at the University of Pittsburgh. IV.D. Lariat formation is not required for efficient splicing in vitro Although branch point attack and lariat formation are strongly preferred events under conditions normally used for self-splicing (Peebles et al., 1986; van der Veen et al., 1986) it has been reported that formation of the lariat is not an essential prerequisite for trans-splicing or self-splicing (Jacquier et al., 1986; Jarrell et al.,1988a; Grivell et al., 1987). I have shown that that a complete deletion of domain 6 (or the branch helix) is with 97 little or no effect on the efficiency of splicing (Figure 51). This proves unequivocally that lariat formation is not critical for in vitro self-splicing. The fact that both the first step (cleavage at the 5* splice-site) and the second step (3* cleavage and exon ligation) in splicing continue to occur efficiently in vitro in the absence of lariat formation (and domain 6 ) raises the question of why group II introns should have maintained that step as part of their splicing mechanism. It is likely that in vivo, nucleophilic attack by the 2*-OH of the branch residue is more efficient than nucleophilic attack by water or 0H-. An alternative explanation is that, in vivo, the intron folds (with the help of proteins) in such a way that water is excluded from the active site, thus favouring lariat formation. IV. E. Domain 5 is also required for the second step in splicing I have clearly demonstrated that domain 5 is required for the first step in splicing by showing that a cis deletion cf domain 5 completely abolishes splicing in vitro. As anticipated, this defect was partially restored when domain 5 function was supplied in trans. Cleavage occured at the 5* splice-site releasing free 5’E and IVS- 3’E. Interestingly, however, the splicing intermediates 5’E and IVS-3'E were unable to carry out the second step in splicing — cleavage at the 3’ splice-site and exon 98 ligation. Similar results were obtained with RNA transcribed from pSH5*PstI and pSH3'PstI (Figure 64). The fact that the absence of domain 5 or the presence of a defective domain 5 in the IVS-3'E precludes 3' cleavage and exon ligation strongly implicates that domain 5 is required for the second step of splicing. It is possible that the absence of domain 5 or the presence of a defective domain 5 impairs its ability to fold into a productive conformation that would make the 3’ splice-site accessible to cleavage. In addition, I have also provided evidence indicating that domain 5 requires an intact domain 3 to carry out the first step in splicing. Several in vivo point mutants have been reported in domain 3 of aI5g (Schmelzer et al., 1982, R. Dietrich, unpublished) that point towards the functional importance of this domain. Detailed structural analysis of domain 3 should help identify regions that are important for in vitro catalysis. IV.E.l. 4 base GAAA loop is not critical for domain 5 function Tuerk et al., (1988) recently reported that loop primary sequences can significantly affect hairpin stability. It has also been suggested that conservation of loop sequences in RNAs indicate sites of protein-nucleic acid or nucleic acid-nucleic acid interactions (Carey et al,, 1983). The fact that mutation of the highly conserved GAAA loop to GAUC in domain 5 did not abolish splicing was 99 rather unexpected. Surprisingly, a drastic alteration (plasmid pSH-Bam 4 ) in which the loop sequence was changed to TCGA and the top helix extended by two base pairs was also with little effect under the high salt conditions. The lack of a strong mutant phenotype maybe because the pSH-Bam HI and the pSH-Bam 4 mutations do not perturb the stability of the helix enough to have an effect on the in vitro reaction. IV.E.2. The 2 base bulge is crucial for domain 5 function Bulged nucleotides, apparently tend to destabilize helical structures. I have shown that a deletion of the 2 base pair bulge in domain 5 completely inactivates the intron. Surprisingly, mutation of the CG bulge to a GC or UC bulge was with little effect on in vitro catalysis suggesting that it is the bulge per se and not the sequence of the bulge that is important. This implies that a certain degree of flexibility in domain 5 structure is required for proper functioning. Perhaps domain 5 undergoes a conformational change during the course of the reaction permitting certain regions that are basepaired in the secondary structure to make crucial tertiary contacts. 100 IV.F. Future experiments IV.F.l. Point of interaction Besults from site-directed mutagenesis (section III.J) indicate that the catalytic region of domain 5 does not depend solely on a single nucleotide or local sequence but may depend on multiple sequences and structural elements arranged by the folding of the RNA. I have shown that the original model proposed by Jarrell (1987) for domain 5 interaction with the 5’ end of the intron is probably incorrect. In vitro analyses of the domain 1 and the Sph I mutation (Section III.J.6) indicate that the current model, proposing an interaction between 7 nts in domain 5 with 7 nts in domain 1, may prove to be correct. Further support is provided by the Tha I point mutant, which is only weakly reactive in vitro suggesting that this nucleotide is critical for the splicing reaction. Additional experiments need to be done to determine whether the effect of the point mutation is due to an unpairing of this nucleotide within domain 5 or due to an unpairing of its interaction with the domain 1 sequence. IV.F.2. Transformation experiments A definitive way of demonstrating that interaction occurs between distant portions of RNA (or protein) molecules is to obtain second-site revertants of mutations that affect function. The point mutants in domain 5 are inactive in the standard and high magnesium splicing buffer 101 and only weakly reactive in the presence of high salt. The conditional nature these mutants implies that in vitro, the conformation of their introns is dependant on ionic strength and composition of the buffer. Now that transformation into yeast mitochondria is possible, analysis of second-site revertants of these and other in vitro mutants should provide important clues to the nature of the interaction. Figure 1. Conserved Sequences in Nuclear, Group I and Group II introns. Nuclear pre-mRNA introns have short conserved sequences at each intron boundary. The 3* terminal AG is always preceeded by a pyrimidine-rich sequence that seperates the highly conserved branch site UACUAAC. These conserved sequences are found in yeast nuclear introns; related sequences are present at those positions in introns of other organisms. Group I introns contain several, short conserved sequence elements (solid boxes). Sequences indicated by the open boxes are not conserved in sequence but always complementary. Sequence elements connected by arrows are capable of pairing. In addition, the IGS is capable of pairing with short 5’ and 3* sequences adjacent to the intron. Group II introns, like nuclear introns, contain conserved boundary sequences. They also contain 2 stem-loop structures at the 3* end of the intron. The one furthest from the 3* end has a highly conserved sequence while the other is not conserved in sequence but contains the lariat branch point. Nuclear pre-tRNAs (not shown) have no consensus sequence at their splice-sites nor are there any conserved sequences within the intron. 102 NUCLEAR pre-mRNA ^ INTRONS UACUAAC / "a GR0UP 1 — A. j f H i l ___CL______INTRONS 1 71 •g s ------* jGUGCG APyj " GROUP II INTRONS Figure 1 Figure 2. Mosaic Genes in Yeast mtDNA. There are 13 introns in most common laboratory strains of Sacchromyces Cerevisiae. Nine are group I (hatched boxes) and four are group II introns (black boxes). Many of the introns contain long ORFs, indicated by the open boxes. 104 105 INTRONS IN YEAST MITOCHONDRIAL GENES o il o I2 oI3 oI4 oI5a oI5/3aC5y OXI * P J1... bI1 bI2 bI3 bI4 bI5 COB □ * Exon/Intron ORF ^ « Group I Intron w+ H * Group H Intron 2IS rRNA f M l 1 M> Figure 2 Figure 3. Transeaterification Mechanism for the Tetrahymena IVS. The 5* and the 3' splice site are denoted by a circle and a square respectively. Exons are indicated by a straight line; wavy lines represent the IVS and C IVS is the covalently closed L-19 IVS. 106 Pr*fftMA 5*----- UCU(g)A ° 0 U - 3' <*0H 0 ®o UCU0 H B u ucuggu Llgat»d txons L IVS G ^ A w UUUqh fG^>A- 4 O C IVS Figure 3 Figure 4. The Internal Guide Sequence (IGS). Lower case letters represent, exons; capital letters, IVS and boxed nucleotides, IGS. Sequences shown are for the Tetrahymena rRNA IVS. The IGS aligns the 5* and 3* exons for splicing. 108 5' splice site \ 5'— u gocucuc u«A AAUAGCAA m i • i t i • « A A AlG 6 6 A G G U U U C C A U U|U A // ------U CG*uao gguogcc 3' splice site Figure 4 Figure 5. The Single "Active Site" or the "Guide Sequence" Model. The self-splicing reaction as well as the subsequent reactions of the excised IVS occur at an oligopyrimidine binding site within the IGS (boxed nucleotides). Conformational changes occur to re-align new tripyrimidine sequences at the binding site until there are no more sequences left to serve as substrates within the active site. n o Ill «c ep- 1* ■« $ • C u egctf A1AUAOCAA.. Pra-rRNA ' • U . I . S J I I J ^ — AAAC G C‘AC GjO UU C C A U U U -----5l -* —■•« 9 • c u c u «e om | Spring Ligottd «»ont •«C«cucucugQO- ^ - 6 AAAUAGCAA. L IVS ...... » i i H cAAAGGGlAGG’UUUCCAUUU* Confo«noltonoi chongt (IroflsftcotioA} CA G A A-U U-A •r" A-U CAAAUACCAAUAUu V a C . t a *^a c c u *u u \ .i J.TJL/G* f A AAGGG-A GGjUU ^A A AjG C GjA G ' | Mojof cyclizotion y Minor cyclizolion C A G A 19-mar U-A C ' IV S ArlJ A AAGGG:AGG'u uu A -0 A A A G G G:A G / « *°A C C O ucu, Oiigonuciaotida binding UCU^fc. c ^AAGGG'AG'g uuu | R«virt« cyclizolion UCU-L* UCUAC. ✓aaaggoagguu Figure 5 Figure 6 . Secondary Structure Model of a Group II intron (aI5g). All group II introns contain highly conserved core secondary structure consisting of a central wheel with six helical "domains" radiating from it (numbered 1- 6 , 112 * « »A. A c A -tT U*AA*U A-U "u * * • A* Ill s*-i 4U*C e © * a- u AUUKS UA, AWCl. u ® V « c=cV X > “\ A V-“ in A •»«CC*oaW’'C* JW cl6. nucl U ■ * m n m • i V © AU& © -•—V A euuese»u,c \a A «U *5U‘» c**.‘ v.\ i#-V fue C £) A*V\t V * A® c1a \ % oUu\u.;«u*uM»‘‘ HAi f V y^ V . “ » „ ca®^' t y ® * u r \ 5 v 4Ue< t f n > ® /V - ueoc >*'em-a*u @ C -/ 33A*y i •a*« Figure 6 Figure 7. Splicing Mechanism of Nuclear mRNA Precursors. Nuclear mRNA precursors splice by a two-step pathway; the first step is cleavage at the 5* splice site resulting in a bipartite intermediate consisting of free 5' exon and the lariat 2/3 RNA. The second step involves cleavage at the 3‘ splice site followed by exon ligation. 114 115 pG U - -A— 4 G j •Up 1: S'tpfleo tAt rtwwjii ♦ I brut tormrtim 1 OH o . ■IT tup): Stpfc* fcN* d H M M A •xan IpatlM I i O k Figure 7 Figure 8 . Comparison of Group I, Group II and Nuclear Intron Splicing. A comparison of all three mechanism shows obvious parallels. As shown, each involves a two step pathway; cleavage at he 5* splice site followed by 3, splice site cleavage and exon ligation. While the two self-splicing introns (group I and group II) involve a transesterification pathway, splicing in nuclear introns is promoted by the splicesome (represented by the large dark circle). 116 117 S«ir-tplicing Group I Group II Nudoar mRNA Cat*}***: RNA RNA anflNP « hnRNP? Maehantam: Q> tniarmxfialK — , , C = 3 < ^ / I I ~ ZS> Br — Q Figure 8 Figure 9. Group II Intron Splicing Pathway. Under the standard and the (NHt^SO* conditions, splicing occurs predominantly by a 2 step transesterification reaction outlined in pathway I. In the presence of KCl, two-thirds of the reacting molecules carry out the alternative first reaction of pathway II; 5* cleavage occurs by hydrolysis rather than by transesterification. The excised (linear or lariat) intron of each pathwayinteracts further with the spliced exons, in KCl, to yield free exons and unaltered intron (spliced exon reopening). iiS 119 I II - ho h n -- O H _ . . P y « , A014 A til----- 1 OAOCO * * d I Transesterification Step 1 I Hydrolysis ioni Release of S’ Exon 1 — 1 i p- Step 2 I Exon Joining 4 .OH .CH Exon Reopening H O - \ , H * tc H | 0H + P C + Intron Figure 9 Figure 10. Plasmid Construction. Plasmid pSH2 was constructed as described in Chapter II (Section II.A.l.). 5’ and 3’ exon are indicated by filled and open rectangles respectively. The line joining the two exons represents the intron (2448 nt). The ORF, that is continuous with upstream exon is represented by the long rectangle (filled). The flanking polylinker sequence and the location of the T3 promoter are shown by stippled and cross hatched areas respectively. The arrow indicates the direction and site of initiation of transcription. Restriction sites relevant to experiments discussed in this dissertation are shown. Sizes of run-off transcripts from plasmid DNA linearized at Cla I, Eco RI and Pvu II are shown. 120 Y fiit Mitochondrial Oxi3-Long g«n« E5 n El E2 E3 E4 a b 9 E8 T-J _____ Lj— _____ I___ ■ ___ I __ H ___ I__ M Hpa II EeoK I I______I pSH2 6.4K b Intron 1 E 2 ORF Hha I S e a l Ace I Cla I Hha I Esolt I Pvu II Template linearized J*so bp wi,h Sizes Of i cia i »" Vitro 3200 b» Runoff ..... — Ee0" 1 Tranacripts ,3<° bp , „ 0 - T3 Promoter w - Polylinker Figure 10 Figure 11. In Vitro Reactivity of the all Precursor RNA. Radiolabeled transcript from Eco RI linearized plasmid pSH2 was reacted for 60 minutes at 45° C in a buffer containing 2 mM spermidine and 10 mM Mg2+ (lane 2), in 100 mM Mg* 4 (lane 3), in 0.5 M (NHthSO* (lane 4), in 0.5 M NH4 Cl (lane 5) and in 0.5 M KCl (lane 6 ). Unreacted precursor RNA is shown in lane 1. 122 Figure 11 Figure 12. Effect of Different Salts on the In Vitro reaction. Radiolabeled precursor RNA was either not reacted (lane 1), or reacted for 60 minutes in various salts of monovalent cations at 0.5 M: LiCl (lane 2), NH«C1 (lane 3), NaCl (lane 4), KCl (lane 5), CHsCOONH* (lane 6), CHaCOONa (lane 7), CHaCOOK (lane 8) and (NIhlaSO* (lane 9). 124 Figure 12 Figure 13. Concentration of Magnesium Chloride. Samples containing radioactive precursor RNA were incubated at 40°C for 2 hours in the presence of 0.5 M NH4CI and varying concentration of magnesium chloride. Lane 1, 0 mM; lane 2, 10 mM; lane 3, 25 mm; lane 4, 50 mM; lane 5, 100 mM. 126 Figure 13 Figure 14. Concentration of NH4 CI. Samples containing radioactive precursor RNA were incubated at 40° C, in a buffer containing 100 mM Mg2+ and varying concentrations of NH4CI. RNA was incubated in the presence of 0 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1 M and 2 M NH4CI in lanes 1-7 respectively. 128 Figure 14 Figure 15. Optimum Reaction Temperature. Radioactive transcripts obtained from plasmid pSH2 cleaved at Eco RI were incubated for 2 hours in 1 H NH4 CI at 0°C, 20° C, 25°C, 30° C, 35«C, 40«C and 45»C in lanes 2-7 respectively. 130 Figure 15 Figure 16. Time Course of the NH4 CI Reaction. Radioactive transcript from plasmid pSH2 linearized at Eco RI was incubated in a buffer containing 100 mM Mg2+ and 1 M NH«C1. Five seperate reactions were incubated at 40° C for 0 min, 10 min, 30 min, 60 min and 120 min (lanes 1-5) respectively. 132 Figure 16 Figure 17. Fate of the downstream exon. Samples containing radiolabeled precursor RNA transcribed from plasmid pSH2 cleaved at Eco RI (lane 3) and at Pvu II (lane 4) were incubated in the all splicing buffer, reactions were stopped after two hours and analyzed. Lanes 1 and 2 are samples of unreacted RNA. 134 135 I 2 3 «h» TXT I IVS-3'E 5'E -3 'E (P v u H ) 5 'E -3 'E it, f>/j .. i 5'E Figure 17 Figure 18. Analysis of 3’ Ends of Precursor and Products. Unlabeled precursor RNA was 3* end-labeled using 5* 32p[pCp] and T4 RNA ligase. A sample of the 3* end-labeled RNA was either unreacted (lane 3) or reacted in the presence of 1 M NHiCl (lane 4). Samples of precursor RNA randomly labeled with 3 2 P UTP were used as controls. Lane 1, precursor RNA; lane 2, reacted precursor RNA. 136 137 12 3 4 IVS-LAR TXT IVS-3t W IVS-BL - 5t -3t 5'E Figure 18 Figure 19. Northern Blot Analysis. Unlabeled reaction products were probed with 5’ end- labeled oligonucleotides, complementary to the 3* exon (lane 2), complementary to the 5* exon (lane 4) and complementary to the intron (lane 6 ). Reaction products in lanes 1, 3 and 5 are randomly labeled at uracil residues and used as markers. 133 TXT — 5 'E -3 'E — INTRON — FRAGMENT s 'E — Figure 19 Figure 20. Primer Extension on 5’E-3*E and 5*E RNAs. Lane 4 contains gel purified S'E-S’E. An end-labeled 18 nt primer complementary to a region of the 3’ exon (See Chapter II) was annealed a sample of the purified S'E-S’E RNA and extended with reverse transcriptase and dNTPs. The product of primer extension is shown in lane 3 (marked by an arrow) and is of the expected size (486 nt). Primer extension was also done (lane 5) on gel purified 5’E RNA (lane 6 ) using a 21 nt 5’ exon specific primer to give a major extension product of the expected size (marked by an arrow). RNAs in lanes 1, 2 and 7 were used as size- standards. 140 Figure 20 Figure 21. Sequencing of Spliced Exons. Purified S’E-S’E was sequenced by the Sanger dideoxy method. An end-labeled 3* exon-specific oligonucleotide was annealed to the 5*E-3*E, extended with reverse transcriptase, unlabeled dNTPS and ddNTPS. The cDNA sequence across the splice junction is shown. N° is an extension reaction in the absence of ddNTPs. 142 Figure 21 Figure 22. 3* End-Labeling of the NH4 CI reaction products. Unlabeled precursor RNA was reacted in 1 M NH 4 Cl and the products 3* end-labeled using 5*3 Zp[pCp] and T4 RNA ligase (lane 2). As a control, internally labeled precursor RNA was reacted in N H < Cl (lane 1) and used as a reference. 144 Figure Figure 23. Debranching Experiment on the NH«C1 IVS-LAR. Purified all IVS-LAR (lane 2) was treated with the debranching extract (lane 3). As shown in lane 3, the IVS- LAR RNA is completely converted to a faster migrating RNA that comigrates with the IVS-BL. As a control, purified aI5g IVS-LAR RNA (lane 5) is shown to debranch (lane 6 ). Lanes 1 and 4 contain the all NH*C1 and the aI5g (NIhhSO* reactions respectively. 146 147 1 2 3 4 5 6 r* Figure 23 Figure 24. Mapping the 5* Ends of the IVS-LAR and IVS-BL. An end-labeled 22 nt intron specific oligonucleotide was annealed to purified IVS-LAR and IVS-BL, extended using reverse transcriptase and unlabeled dNTPs. The products were fractionated alongside a DNA sequencing ladder. As shown both the IVS-LAR (lane 1) and IVS-BL (lane 2) yield the same 192 nt primer extension product. 148 149 LADDER m m 192 bc Figure 24 Figure 25. Concentration of KC1 Addition of KC1 promotes the appearance of novel products. Radiolabeled transcripts were incubated for 2 hours with 100 mM Mg2* and 50 mM, 100 mM, 250 mM, 500 mM and 1 M KC1 (lanes 1-5 respectively). Lane 6 contains unreacted RNA. 150 Figure 25 Figure 26. Time Course of the KC1 Reaction. Radioactive precursor RNA from Eco RI linearized plasmid pSH2 was incubated for various times in the reaction buffer containing 100 mM Mg 2 + and 1 M KC1. Reactions were incubated at 40° C for 2, 5, 10, 20, 30, 45, 60, 120 min, lanes 1 - 1 0 , respectively. 152 Figure 26 Figure 27. Mapping the 5* ends of the Novel Linear RNAs in KC1. Primer extensions were done on the novel linear RNAs using a 22 nt primer, complementary to a sequence 170-192 nt from the 5'end of the intron. Products were fractionated alongside a DNA sequencing ladder. As shown both the 520 nt (lane 1) and the 262 nt RNA (lane 2) yield a 192 bp extension product that maps exactly to the 5' end of the intron. 154 LADDER Figure 27 Figure 28. Gel Purification and Re-electrophoresis of the Novel Non-Linear RNA Species in KC1. The two slow moving species in KC1 were gel purified (lane 4) and reacted in the presence of 1 M KC1 (lane 5). They were fractionated next to unreacted precursor RNA (lane 1), NH4 CI and KC1 reaction products (lanes 2 and 3 respectively). 156 TXT IVS-BL Figure 28 Figure 29. 3* End-Labeling of Non-Linear RNAs in KC1. Unlabeled precursor RNA was reacted in 1 M KC^ and the reaction products 3’ end-labeled with 5'3ap[pCp] and T4 RNA ligase (lane 2). In lane 1, KC1 reaction products of internally labeled precursor RNA were used as a reference. 158 IVS-LARs — 5'£-3'E — mm INTRON — FRAGMENT Figure 29 Figure 30. Proposed Reaction Pathway in KC1. The overall splicing mechanism is similar to aI5g. Cleavage at the 5* splice site occurs by hydrolysis rather than by transesterification. This followed by cleavage at the 3* splice site and exon ligation to yield spliced exons and excised full-length linear intron. 160 Step t Hydrolysis Release, of .5' Exon l Step 2 EaftpJsining. Transesterification Spliced Exons 5 ’ OH 3’ P™ A — o » U n e ,r Intron Figure 30 Figure 31. Proposed Model for the Post-Splicing Reaction in KC1. In this model, the excised full-length linear intron is capable of cleaving itself at either of two sites near its 5* end yielding 5’ intron fragments and IVS-LARs smaller than the full-length IVS-LAR. 162 Linear OH Intron Q. Q 5’ Intron Lariats Fragments Figure 31 Figure 32. Evidence for the Post-Splicing Reaction. Gel purified IVS-LIN RNA either not reacted (lane 1), reacted in NH«C1 (lane 2) and KC1 (lane 3). Products of the post splicing reactions were fractionated alongside all NH4 CI and KC1 splicing reactions (lanes 4 and 5 respectively. While the IVS-LARs and the larger intron fragment are clearly visible in lane 3, the smaller intron fragment was runoff the gel. 164 165 2 3 4 5 IVS-LAR TXT IVS-BL * ♦ 5"E - 3 'E INTRON FRAGMENT m 5'E Figure 32 Figure 33. All Does Not Carry Out Spliced exon reopening. Purified all 5*E-3*E was incubated for 1 hour in the KC1 splicing buffer (lane 2) and in the presence of KC1 plus an excess (30 fold) of unlabeled precursor RNA (lane 3). As a control, purified aI5g 5*E-3'E was incubted in KC1 (lane 5) and in KCl plus an excess (30 fold) of its unlabeled precursor RNA (lane 6). Lanes 1 and 4 contain the all and the aI5g splicing reactions in KCl. 166 167 2 3 4 5 6 TXT (II) TXT (159) 5'E-3'E — 5t-3fe 5"e 3% 5 " E Figure 33 Figure 34. Heterologous SER experiment. Purified all 5'E-3’E was incubated in KCl (lane 1), in KCl plus an excess of unlabeled all precursor RNA (lane 3) and in KCl plus an excess of unlabeled aI5g precursor RNA (lane 4). While aI5g 5*E-3’E was reacted in KCl (lane 5), in KCl plus an excess of unlabeled aI5g precursor RNA (lane 6) and in KCl plus an excess of unlabeled all precursor RNA (lane 7). Unlabeled precursor RNA in all these reactions was 30 fold in excess. 168 169 12 34 5 6 7 Figure 34 Figure 35. In Vitro Reactivity of the Shortened all Intron. Radioactive RNA transcribed from plasmid pSHAAC linearized at Eco RI (lanes 7-12) and full-length precursor RNA (lanes 1-6) were reacted under comparable reaction conditions. RNAs were unreacted in lanes 1 and 7, reacted in 2 dM spermidine and IQ mM MgCl2 (lanes 2 and 8), in 100 mM MgClz (lanes 3 and 9), in 1 M (NH«)2 SO< (lanes 4 and 10), in 1 M NHtCl (lanes 5 and 11) and in 1 M kCl (lanes 6 and 12). 170 171 I 2 34 5 £ 78 9 10 II 12 Figure 35 Figure 36. Treatment of the Shortened IVS-LAR RNA with the Debranching Extract. IVS-LAR RNA was purified from the reaction products of the shortened precursor RNA (plasmid pSHAAC) and incubated in the absence and in the presence of the debranching enzyme (lanes 5 and 6). As a control, purified aI5g IVS-LAR RNA in lane 2 is shown to be debranched by the S100 extract (lane 3). Lanes 1 and 4 contain the aI5g and the shortened all splicing reactions in (NH«)2 S0 4 and NH«C1 respectively. 172 173 I Z 3 4 5 6 m dMH — IVS-LAR IVS-LAR ----- — TXT TXT ----- ■SCWfRT’ -IVS-BL I VS - b l ------».<■' c *5E-3'E 5 W E _ -5% Figure 36 Figure 37. Limit T1 Digest on the IVS-LAR and the IVS-BL RNA. 3* end-labeled IVS-LAR (lane 2) and IVS-BL RNA (lane 3) were digested to completion with RNase Tl. The products were fractionated on a 25% polyacrylamide gel. 5* end- labeled oligodeoxyribonucleotides (lane 1) were used as rough size standards. 174 175 2 3 'J Figure 37 Figure 38. Schematic Representation of the "Half Molecules" used in the Trans-Splicing Reactions. As shown in the schematic, transcripts constituting the upstream "half molecules" contain 5’ exon, part of the intron containing domains 1-3 plus varying amounts of domain 4 (lines 2-5). The downstream "half molecule" (transcribed from Eco RI linearized plasmid pSH4) contains intron sequences downstream of the unique Cla I site and the 3’ exon. Line 1 represents the full-length precursor RNA with intron domains indicated by the numbered rectangles, while line 6 shows the shortened intron form. 176 DELETION SPLICED EXONS 5' Exon y Exon Hi_ + “ 1 3 Cll I | He®* t Cl» I + * i — u EcoH 1 T»q I n 7«7nt + Eco* I n 1 1 0 7 n l 4- • E coH I I Un 158*nt + Eeo* I a 1107nt + Eeo* I Figure 38 Figure 39. All Precursor RNA Interrupted Within Domain 4 Trans-Splices In Vitro. In this experiment radiolabeled transcript transcribed from Cla I linearized plasmid pSH2 constituted the upstream half molecule while unlabeled RNA transcribed from Eco RI linearized plasmid pSH4 constituted the downstream half moldcule. The Cla I run-off transcript was mixed with an excess of the unlabeled Cla I to Eco RI RNA, but not not incubated (lane 3), incubated in NH4 CI (lane 5) or in KCl (lane 5). Lanes 1 and contain the full-length precursor RNA in KCl and NH 178 179 IVS-LAR TXT CI clI r u n o ff I VS-Cl 0.1 S'E-3'E in tr o n fr a g m e n t . ...» t , ^ ■ $\k f I , 5'E ■>#•» V £ \ l t r Figure 39 Figure 40. Most of Domain 4 is not Required for In Vitro Trans-Reactions. Radioactive RNA from pSH2 linearized at Acc I (lanes 3-5) and Sea I (lanes 6-8) were mixed with an excess of the unlabeled pSH4 RNA. Samples of the mixed RNAs were either not incubated (lanes 3 and 6), or were ncubated in the presence of 1 M NH« Cl (lanes 4 and 7) or 1 m KCl (lanes (5 and 8). RNA in lanes 1 nad 2 is the full-length precursor reacted RNA#in NH«C1 and in KCl respectively. 180 181 I 2 3 4 5 67 8 IVS-LAR TXT 5fe-3'E in tr o n - fr a g m e n t 5"e INTRON - FRAGMENT Figure 40 Figure 41. The Conserved Boundary Sequence is Required for Trans-splicing. RNA transcribed from plasmid pSH3 was mixed with an excess of purified 5* E. Samples were either not incubated (lane 4) or incubated in NH« Cl (lane 5). As controls, purified 5'E RNA and RNA from pSH3 were either not incubated (lanes 2 and 6 respectively), or incubated in NH«C1 (lanes 3 and 7 respectively). Lane 1 contains precursor RNA reacted in NKU Cl . 182 Figure 41 Figure 42. Sequence Analysis of Domain 5 Deletion. Domain 5 deletion (Plasmid pSHA5) was confirmed by double strand DNA sequencing. The region spanning the deletion is merked by an asterisk. 184 Figure 42 Figure 43. Domain 5 is Required for the Second Step in Splicing. (A) RNA from pSHA5 was either not incubated (lane 3) or incubated in 2 mM spermidine and 10 mM MgCla (lane 4), in 100 mM MgCla (lane 5), in 0.5 M (NH«)2S0« (lane 6) and in 0.5 M KCl (lane 7). RNA in lanes 1 and 2 is wildtype precursor RNA in (NHi^SO* and KCl respectively. (B) A sample of the pSH 5 RNA was not incubated (lane 6). This RNA was mixed with an excess of unlabeled IVS 5 RNA (plasmid pJDI5’-75) and was either not reacted (lane 7) or reacted in 0.5 M KCl (lane 8). While lanes 1 and 2 contain wild type RNA in (NH4 )2 S0 4 and KCl respectively, lanes 3-5 are control lanes containing RNA transcribed from plasmid pJD20-Hae III. Controls lanes were incubated in exactly the same way as described for lanes 5-8. 18c Figure 43 Figure 44. Domain 3 is required for 5* cleavage. RNA transcribed from plasmid pJD20-Xba I linearized at Xba I was either not reacted (lane 5) or reacted in KC1 (lane 6) or mixed with an excess of unlabeled IVS 5 RNA and reacted in KC1 (lane 7) . In lanes 2-4 the experiment was done exactly the same way as in lanes 5-7, except that RNA from pJD20-Hae III was used instead of pJD20-Xba I RNA. Wild type precursor RNA reacted in KCl is shown in lane 1. 188 189 I 2 3 4 5 6 7 IVS-LAR TXT IV 5 -B L 3 ‘*E 5'E Figure 44 Figure 45. Proposed Model for Domain 5 Interaction. 5’ half of the bottom helix of domain 5 can potentially base pair with the first 7 nt of the intron. Exons are represented by rectangular boxes while the intron domains are are numbered 1-6, 5*-3*. 190 191 ^•5 <1 p "2 A ^ 3 ouuuuc/GUGCGGU GCCGUAUG Figure 45 Figure 46. The FIB IVS-LABs are not Susceptible to the Debranching Extract. The FIB IVS -LARs were purified and samples were either not incubated (lanes 2 (wild type), 5 (FIB C), 8 (FIB A) and 11 (FIB U).or incubated in the presence of the debranching activity (lanes 6 , 9 and 12). Lanes 1, 4, 7 and 10 contain the wild type and the FIB C, A and U mutant RNAs reacted in ( NH4 ) 2 SO4 respectively. 192 193 2 3 4 5 6 7 8 9 |0 H \ z IVS-LAR- TXT - « 1VS-BL— * S'E-^E- *»» m * Figure 46 Figure 47. Sequence Analysis of 5* intron Pst I Mutation. Insertion of the Pst I site at the 5’ end of the intron (by site-directed mutagenesis) was confirmed by double strand DNA sequencing. The region spanning the mutation is marked by an astrisk. 194 195 Figure 47 Figure 48. In Vitro Analysis of pSH5’I-Pst I RNA. RNA transcribed from plasmid pSH5'I-Pst I was not incubated (lane 1), incubated in 100 mM MgClj (lane 2), in 0.5 M (NH4 )2 S0 4 (lane 3) and in 0.5 M KC1 (lane 4). 196 Figure 48 Figure 49. pSH5*I-Pst I RNA is blocked in SER. Lanes 1 and 2 contain wild type IVS-LIN and 5’E-3’E RNA, lanes 3 and 4 wild type IVS-LIN RNA and 5*E-3'E from pSH5»Pst I RNA, lanes 5 and 6 pSH5’I~Pst I IVS-LIN and 5*E- 3'E RNA and lanes 7 and 8 pSH5’I-PstI IVS-LIN RNA and wild type 5'E-3'E. RNAs were mixed in roughly equimolar quantities and either not incubated (lanes 1, 3, 5, and 7 ) or incubated in 0.5 M KC1 for 2 hours at 45 °C (lanes 2, 4, 6 and 8 ) 198 Figure 4S Figure 50. Sequence Analysis of Domain 6 Deletion. Domain 6 deletion was confirmed by sequencing plasmid pSHA6 by double strand DNA sequencing. 200 Figure 50 Figure 51. Domain 6 is not Required for Splicing In Vitro. RNA transcribed from plasmid pSH A 6 was analyzed in vitro. The RNA was either unreacted (lane 1) or reacted in 2 mM spermidine and 10 mM MgCl2 (lane 2), in 100 mM MgCl2 (lane 3), in 0.5 M (NH«)2 S0 4 (lane 6 ) and in 0.5 KC1 (lane 7). RNAs in lane 1 and 2 are wild type precursor RBA reacted in (NH4 )2 SO« and KC1 respectively. 202 203 12 3 4 5 6 7 IVS- l AR TXT 5'E-3'E 3'E 5 E Figure 51 Figure 52. Structure of Domain 5. The sequence (5'-3 *) and the secondary structure of domain 5 is shown. It consists of a highly conserved, 34 nt stem-loop structure with a 4 base loop and a 2 base bulge. 204 205 A A G A U G A - U G =C C = G G = C U - A G G A - U U -A G = C C = G C = G G . U A-U Figure 52 Figure 53. Comparison of all and aI5g domain 5. The boxed stretches and nucleotides are conserved between the two intron. As shown the two structures are very similar and only differ at 9 positions. 206 207 DOMAIN 5 A A 6 A U G A - U U-A G = C C *= G G = C U-A C C G fA-U^ A - U U-A U - A 6 *C G = C C a= G C = G C «G C as G G . U G . U lA-uJ A - U 5* A - U - ■ 3’ 5’- -G»C- - 3 * ah al5g Figure 53 Figure 54. Domain 5 of AI5g Cleaves At or Near the 5 Splice Site of All. Cla I run-off transcript from plasmid pSH2 was either not reacted (lane 1), or reacted in KC1 (lane 2). The RNA was mixed with an excess of unlabeled aI5g domain 5 RNA and incubated in 1 M KC1 for 2 hours at 40 °C (lane 3). RNA in lane 4 is all precursor RNA reacted in KC1. 208 CICLl - RUNOFF Figure 54 Figure 55. Sequence Analysis of the Bam HI and the Bam-4 Mutation in Domain 5. In panel (A) the sequence of the plasmid pSH-Bam HI is shown and the region of the Bam HI mutation is marked by an asterisk. In panel (B) region of the Bam-4 mutation is indicated by an asterisk. 2L0 211 Figure 55 Figure 56. In Vitro Analysis of the Bam HI and the Bam-4 RNA. RNA transcribed from plasmid pSH-Bam HI (lanes 8-12) and plasmid pSH-Bam4 (lanes 3-7) were analyzed in vitro. RNAs were not reacted (lanes 3 and 8 ), reacted in 2 mM spermidine and 10 mm MgCl2 (lanes 4 and 9), in 100 mM MgCla (lanes 5 and 10), in 0.5 M ( N H O 2 SO4 (lanes 6 and 11) and in 0.5 M KC1 (lanes 7 and 12). RNAs in lanes 1 and 2 are wild type precursor RNA reacted in (NH«)2 SO« and in KC1 respectively. 212 213 I 2 4 5 £ 7 8 9 10 II 12 IVS-LAR TXT 5 E - 3 t 3'E s' e <*>& Figure 56 Figure 57. Schematic representation of the run-off experiment. Line 1 represents precursor RNAs truncated at Hpa II ( pJDI3’-851), line 2 represents an Rsa I run-off transcript from plasmid pJD2 while line 3 represents a Bam HI run-off transcript from plasmid pSH-Bam HI. 214 215 5 EXON RELEASE o 5E 3E Hpall + R 2 JSS. Rsa. Figure 57 Figure 58. RNA Truncated at Rsa I Does Not Release free 5 ’exon. RNA transcribed from pJDI3*-851 (lanes 4-6), Rsa I run off transcript (lanes 7-8) and Bam HI run-off trancript (lanes 10-12) were analyzed in vitro. RNAs were not reacted (lanes 4, 7 and 10), reacted in 0.5 M (NH«)aSO« (lanes 5, 8 and 11) and in 0.5 M KC1 (lanes 6, 9 and 12). RNAs in lanes 1-3 are wild type precursor RNA either unreacted or reacted in (NH<)2SO« and KC1 respectively. 216 217 1 2 3 4 5 6 7 8 9 10 II |2 IVS-LAR- TXT- 5 c - 3 t 3"E 5'E Figure 58 Figure 59. Sequence Analysis of the 2 base Bulge.Mutants The mutants were confirmed by DNA sequencing. Panel (A) shows the sequence of the CG deletion mutant, panel (B) the sequence of the GC mutant and panel (C) the UC mutant. In all three panels, the region pertaining to the mutation is marked by an asterisk. 218 § ° t> * # + n I 11 ii III ii 11 I n f I I III! I l l • f ° • • litt«H» • ? « * i ii tit I M MI itit * > i ii Figure 59 6TZ Figure 60. Xn Vitro Analysis of the Bulge Mutants. In panel (A) RNA was transcribed from plasmid pSHACG (lanes 1-5), in (B) from plasmid pSH-GC (lanes 1-5) and in (C) from plasmid pSH-UC (lanes 1-5). In all three panels RNAs were not reacted (lane 1), reacted in 2 mM spermidine and 10 mm MgCls (lane 2), in 100 mM MgCl2 (lanes 3), in 0.5 M (NH«)2 SCU (lane 4) and in 0.5 M KC1 (lane 5). 220 Figure 60 Figure 61. Schematic Representation of Mutations in the Bottom Helix of Domain 5. The sequence of domain 5 in the pSH5’Pst I and the pSH3’Pst I RNA are shown. Wild type sequence is represented by lower case and the mutant sequences by upper case letters. 222 a a 0 a 0 a u . g u . g a — u a — u 8 “ c 0 - C c -g e-0 g- e g - c u -a u - a e c 0 O G . u a C A a u U C c 0 G G g c C U . g e A C u 0 G 5'E 3 'E 5'E a - u a - u 3'E -ih -g -c------~ih _0»c-- JU DOM AIN 5 DOM AIN 5 ( S’ Pat mutation ) ( 3* Pat mutation ) Figure 61 223 Figure 62. Sequence Analysis of the Bottom Helix mutants. The sequence of the plasmid pSH5'PstI (A) and plasmid pSH3*PstI (B) confirmed by DNA sequencing. The region in which the mutation resides is marked by an asterisk. 224 225 Figure 62 Figure 63. In Vitro Analysis of the Bottom Helix mutants. RNA transcribed from plasmid pSH5*Pst I (lanes 3-7) and plasmid pSH3'PstI (lanes 8-12) were analyzed in vitro. RNAs were not reacted (lanes 3 and 8), reacted in 2 mM spermidine and 10 mm MgCl2 (lanes 4 and 9), in 100 mM MgCl2 (lanes 5 and 10), in 0.5 M (NH<)2S0« (lanes 5 and 11) and in 0.5 M KC1 (lanes 7 and 12). RNA& in lanes 1 and 2 are wild type precursor RNA reacted in (NHOjSO* and in KCl respectively. Figure 63 Figure 64. Domain 5, in Trans, Partially Supresses the Mutant Phenotype of the Bottom Helix Mutants. Samples of pJD20-Hae III, pSH5’Pst I and pSH3‘PstI RNA were either not reacted (lanes 3, 6 and 9), reacted in 0.5 M KC1 (lanes 4, 7 and 10) or incubated with an excess of IVs 5 RNA (lanes 5, 8 and 11). RNAs in lanes 1 and 2 are wild type precursor RNA reacted in (NH4 )2 SO« and in KC1 respectively. 228 229 1 2 3 4 5 6 7 8 3 10 Ii IVS-LAR- TXT - —TXT 5"E-3'E s'e Figure 64 Figure 65. Working Model for Domain 5 Interaction. Seven nt in the 5* half of domain 5 can form a perfect match with seven nt in domain 1 . 230 EBSI A A G A U G A-U A G = C U G *c G = C C = G U - A G = C A-U U-A fi> fi> O A - U 3* U - A G = C C = G C = G G . U A-U 5’ — G = C — DOMAIN 1 DOMAIN 5 Figure 65 Figure 66. Point Mutants in Domain 5. The sequence around the point mutants in domain 5 is shown. Nucleotides that were altered by site-directed mutagenesis are indicated by arrows. 232 Point mutants C = G G = C 1. U-A C G A-U U-A G = C Wild type (domain 5) Figure 66 Figure 67. Sequence Analysis of the point mutants in domain 5. The point mutants pSH-Sphl (B) and pSH-Thal were confirmed by DNA sequencing. The region in which the point mutants were inserted is indicated by an asterisk. 234 Figure 67 Figure 68. In Vitro Analysis of the Point Mutants. RNA transcribed from plasmid pSH-Thal (lanes 3-7) and plasmid pSH-Sphl (lanes 8-12) were analyzed in vitro. RNAs were not reacted (lanes 7 and 12), reacted in 2 mM spermidine and 10 mm MgClz (lanes 6 and 11), in 100 mM MgCl2 (lanes 5 and 10), in 0.5 M (NHU^SO* (lanes 4 and 9) and in 0.5 M KCl (lanes 3 and 8 ). RNAs in lanes 1 and 2 are wild type precursor RNA reacted in (NH«)2 SCU and in KCl respectively. 236 IVS -LAR TXT 5t-3"E 5 t Figure 68 Figure 69. Sequence Analysis of the Domain 1 Mutation. The sequence of the mutant pSH-Doml was confirmed by DNA sequencing and the region pertaining to the mutation is indicated by an asterisk. 238 Figure 69 Figure 70. In Vitro Analysis of the Domain 1 mutant RNA. RNA transcribed from plasmid pSH-Doml (lanes 3-7) and was analyzed in vitro. RNA was not reacted (lane 1), reacted in 2 mM spermidine and 10 mm MgClz (lane 2), in 100 mM MgCl2 (lane 3), in 0.5 M (NH4 )2 SO« (lanes 4) and in 0.5 M KCl (lanes 5). 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