Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Phosphorylation and functions of the RNA polymerase II CTD Hemali P. Phatnani1 and Arno L. Greenleaf2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA The C-terminal repeat domain (CTD), an unusual exten- important that the heptads be in tandem: Insertion of an sion appended to the C terminus of the largest subunit of Ala residue between heptads is lethal in yeast, whereas RNA polymerase II, serves as a flexible binding scaffold insertion of an Ala between heptad pairs can be tolerated for numerous nuclear factors; which factors bind is de- (Stiller and Cook 2004). While the CTD is indispensable termined by the phosphorylation patterns on the CTD in vivo, it is frequently not required for general transcrip- repeats. Changes in phosphorylation patterns, as poly- tion factor (GTF)-mediated initiation and RNA synthesis merase transcribes a gene, are thought to orchestrate the in vitro (Zehring et al. 1988; Kim and Dahmus 1989; association of different sets of factors with the transcrip- Buratowski and Sharp 1990; Kang and Dahmus 1993; tase and strongly influence functional organization of Akoulitchev et al. 1995). Thus, the CTD does not form the nucleus. In this review we appraise what is known, part of the catalytic essence of RNAPII; rather, it must and what is not known, about patterns of phosphoryla- perform other functions. The nature and variety of these tion on the CTD of RNA polymerases II at the begin- functions are currently being elucidated and are a main ning, the middle, and the end of genes; the proposal that topic of this review. doubly phosphorylated repeats are present on elongating A feature of the CTD that was discovered early, and polymerase is explored. We discuss briefly proteins that clearly carries functional implications, was that it is known to associate with the phosphorylated CTD at the subject to hyperphosphorylation. RNAPII can exist in a beginning and ends of genes; we explore in more detail form with a highly phosphorylated CTD (subunit II0; proteins that are recruited to the body of genes, the di- RNAPII0) and a form with a nonphosphorylated CTD versity of their functions, and the potential conse- (subunit IIa; RNAPIIA) (for a review, see Dahmus and quences of tethering these functions to elongating RNA Dynan 1992). Phosphorylation occurs principally on Ser2 polymerase II. We also discuss accumulating structural and Ser5 of the repeats (Dahmus 1995, 1996), although information on phosphoCTD-binding proteins and how these positions are not equivalent (West and Corden it illustrates the variety of binding domains and interac- 1995; Yuryev and Corden 1996). A consequence of hy- tion modes, emphasizing the structural flexibility of the perphosphorylation is that the mobility in SDS gels of CTD. We end with a number of open questions that the II0 form of the largest subunit is markedly reduced highlight the extent of what remains to be learned about relative to that of form IIa (e.g., see Greenleaf 1992). the phosphorylation and functions of the CTD. Learning that RNAPII could exist in two forms led to efforts to understand functional differences between them. We now know that the phosphorylation state The C-terminal repeat domain (CTD) of RNA polymer- changes as RNAPII progresses through the transcription ase II (RNAPII) is an amazing sequence arrangement at cycle. the end of the largest RNAPII subunit (apologies to Early results from Dahmus suggested that the initiat- Chow et al. 1977). This “domain” is inherently unstruc- ing RNAPII was form IIA while the elongating enzyme tured yet evolutionarily conserved, and in fungi, plants, was form II0 (Cadena and Dahmus 1987; Payne et al. and animals it comprises from 25 to 52 tandem copies of 1989). In the meantime, the first CTD kinase (yeast the consensus repeat heptad Y1S2P3T4S5P6S7 (Corden CTDK-I) was purified (Lee and Greenleaf 1989, 1991; 1990). The CTD is essential for life: Cells containing Sterner et al. 1995) and used to prepare biochemical only RNAPII from which two-thirds or more of the re- amounts of hyperphosphorylated recombinant CTD, peats have been removed are inviable (Nonet et al. 1987; which was then employed to generate and affinity purify Zehring et al. 1988; for a review, see Corden 1990). It is antiphosphoCTD antibodies (Lee and Greenleaf 1991; Weeks et al. 1993). These antibodies were used in fluo- [Keywords: CTD; RNA polymerase II; cotranscriptional; nuclear organi- rescence microscopy to investigate the in vivo distribu- zation; phosphorylation] 1Present address: Department of Molecular and Cellular Biology, Har- tion of RNAPII0 on Drosophila polytene chromosomes. vard University, Cambridge, MA 02138. Consistent with the results from Dahmus, this approach 2Corresponding author. E-MAIL: [email protected]; FAX (919) 684-8885. demonstrated that sites of active transcription contained Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1477006. RNAPII0, whereas some inactive genes and promoter- 2922 GENES & DEVELOPMENT 20:2922–2936 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press PhosphoCTD of RNA polymerase II proximal sites with paused polymerases contained and Reed 2002; Proudfoot et al. 2002). We now under- RNAPIIA (Weeks et al. 1993). Using a different cross- stand that the PCTD, via its recruitment of PCTD-bind- linking method and assay, Lis and colleagues (O’Brien et ing factors, plays a major role in coordinating a number al. 1994) observed the same distribution. These results of nuclear processes with RNA chain synthesis and the reinforced the idea that promoter binding and early translocation of RNAPII along a gene. events are carried out by RNAPIIA, whereas elongation The role of CTD phosphorylation in facilitating pre- is carried out by RNAPII0 (Dahmus 1994). Almost all mRNA processing has thus far been best characterized subsequent experiments are consistent with this overall for 5Ј-end capping and 3Ј-end cleavage and polyadenyla- notion. However, it should be kept in mind that some tion. The 7-methyl G5Јppp5ЈN cap is added when the genes may differ from this picture (e.g., Lee and Lis transcript is ∼25 bases long, soon after its 5Ј end emerges 1998). An example of gene class-specific differences in from the exit channel of RNAPII (Jove and Manley 1984; CTD phosphorylation was already found in 1993: By im- Rasmussen and Lis 1993). That acquisition of such a cap munofluorescence, elongating RNAPs on developmen- is unique to RNAPII transcripts (Shatkin 1976), and tran- tally induced loci in Drosophila (ecdysone puffs on poly- scripts made by a CTD-less RNAPII are very ineffi- tene chromosomes) were recognized exclusively as “II0” ciently capped (McCracken et al. 1997a), suggested that enzymes, whereas RNAPs on stress-induced loci (heat- capping enzyme associates with the transcription com- shock puffs) were recognized as both “II0” and “IIA” plex via interactions with the CTD. An exploration of forms (Weeks et al. 1993). this hypothesis led to the finding that capping enzyme It is very important to note that the “II0” designation indeed associates physically with the CTD of RNAPII in simply indicates hyperphosphorylation of the CTD (as vitro (Cho et al. 1997; McCracken et al. 1997a). Subse- detected originally by mobility shift of the Rpb1 sub- quent cross-linking studies (chromatin immunoprecipi- unit); RNAPII0, however, is not necessarily a homoge- tation, or ChIP) showed that capping enzyme also asso- neous population of molecules. While RNAPII0 does ciates with transcribed genes in vivo, in a manner that consist of RNAPs with hyperphosphorylated CTDs, the requires CTD phosphorylation; consistent with 5Ј cap- patterns of phosphorylation on individual CTDs can ping being an early event in the life of a nascent tran- vary widely. This variation can be due to differential script, capping enzyme localizes to genes near their 5Ј phosphorylation of Ser2 versus Ser5 residues and/or to ends (Komarnitsky et al. 2000; Schroeder et al. 2000). differential phosphorylation of repeats along the length Analogous to capping, the formation of 3Ј ends of mes- of the CTD. As expanded on below, modulating these sages is also coupled to transcription by RNAPII through patterns regulates the affinity of the CTD for its binding interactions between the CTD and the processing ma- partners, and consequently different phosphorylation chinery (for a review, see Proudfoot 2004). Attempts to patterns present at different stages of transcription con- uncover the biochemical basis of this functional link led trol the timely recruitment to transcribing RNAPII of to the finding that cleavage and polyadenylation factors factors important for RNA maturation and other events. bind to the PCTD in vitro (McCracken et al. 1997b; Birse Much of this review deals with what these CTD phos- et al. 1998). ChIP experiments reveal that CF IA, a factor phorylation patterns may be, how they are created, and involved in 3Ј-end formation, accumulates toward the 3Ј what their functional significance is. While the recent ends of genes (Licatalosi et al. 2002), and its cross-linking past has witnessed significant progress toward answering is dependent on CTD phosphorylation (Licatalosi et al. these questions, our hope is that this review will under- 2002; Ahn et al. 2004). score the point that we have a great deal to learn about virtually every aspect of CTD phosphorylation and func- CTD phosphorylation patterns along genes tion. With the advent of ChIP it became feasible to explore the Patterns and consequences of CTD phosphorylation phosphorylation status of the CTD on RNAPs at differ- ent positions along a transcription unit. The commercial The CTD as a binding scaffold: linking nuclear availability of anti-CTD monoclonal antibodies (mAbs) processes to transcription with phosphorylation pattern-dependent specificities If the CTD is not required for catalyzing the synthesis of helped spur these studies.