The RNA polymerase II CTD coordinates transcription and RNA processing

Abstract

The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity.

Figure 1.

Comparison of select CTD sequences. The CTD sequences of fission and budding yeast, zebrafish, and humans are shown and aligned to display the context of the heptad repeats. All-consensus YSPTSPS repeats heptads are in red, and the numbers next to the parentheses indicate the repeat number. The CTD of fission yeast contains 29 heptads, 24 of which are all-consensus, whereas the CTD of budding yeast consists of 26 heptads, 19 of which are perfect consensus. The fish and human CTDs each consist of 52 repeats, with a 10- to 11-residue motif (in bold) at the very C terminus. Highlighting the conservation of the CTD among vertebrates, fish and human CTDs are 97% identical. Residues in the fish CTD that differ from humans are in yellow, and human residues that deviate from the consensus are in blue.

Figure 2.

Dynamic modification of the CTD during the transcription cycle. At transcription initiation, CDK7 phosphorylates Ser5 and Ser7 residues. Later, during elongation, CDK9 phosphorylates Ser2 and perhaps Thr4, while the phosphate groups on Ser5 and Ser7 are gradually removed by phosphatases. For example, Rtr1, likely indirectly with another phosphatase, and Ssu72, with the aid of the prolyl isomerase Pin1, dephosphorylate Ser5-P early and late during elongation, respectively. Ssu72 also dephosphorylates Ser7-P. CDK12 likely also contributes to Ser2 phosphorylation during elongation of at least some genes. As RNAP II nears termination, Fcp1 dephosphorylates Ser2-P, regenerating unphosphorylated RNAP II that can be recycled for another round of transcription.

Figure 3.

The CTD facilitates capping and splicing by recruitment of RNA processing factors. (A) Capping enzyme (CE) is recruited to the vicinity of nascent mRNA by the CTD phosphorylated on Ser5. (B) During transcription, the CTD is phosphorylated on Ser2, while the Ser5-P is dephosphorylated and is involved in recruiting the indicated splicing factors, which defines splice sites and facilitates assembly of the spliceosome. In this and subsequent figures, green spheres above the CTD represent relevant CTD-binding proteins, while assembled functional complexes are indicated below.

Figure 4.

The CTD functions in 3′ processing of both polyadenylated and nonpolyadenylated RNAs. (A) At 3′ ends of polyadenylated mRNA, Ser2-P serves to recruit Pcf11, a component of CFII (human nomenclature is used for all factors). Other 3′ end factors, such as CstF50 and AAUAAA-binding factor CPSF-160 (Yhh1 in yeast), also bind the CTD, whereas Ssu72, with the aid of Pin1 (Ess1), must dephosphorylate Ser5-P. The AAUAAA element and G/U elements are bound by CPSF and CstF, respectively. Loading of some factors, including CPSF and CstF, may occur upstream, perhaps at the promoter (see the text). (B) The 3′ end of histone pre-mRNA contains a stem–loop motif bound by SLBP and a downstream element recognized by U7 snRNP. A complex containing CPSF73, CPSF100, and Symplekin is recruited for 3′ cleavage. Thr4-P facilitates this process, likely through a yet-to-be-identified factor. (C) The 3′ end of snRNA genes contains a 3′ box that interacts with the Integrator complex. RPAP2 binds to Ser7-P on the CTD and to recruit the Integrator, and Int 11, an Integrator subunit, cleaves the RNA.

Figure 5.

The CTD facilitates different termination mechanisms for protein-coding and noncoding genes. (A) Poly(A)-dependent termination pathway. RNA is cleaved by 3′ end processing factors at the polyadenylation site. The CTD with Ser2-P is involved in recruiting factors, including Pcf11, Rtt103, p54/PSF, and Sen1, to facilitate termination of long polyadenylated transcripts. Pcf11 and Rtt103 are required for the recruitment of exoribonuclease Rat1 in yeast, while Xrn2 is recruited by p54/PSF in humans. Sen1 (Senataxin in humans) may function on some of these genes by resolving RNA–DNA hybrids. (B) Nrd1c-dependent termination pathway. The Nrd1 complex (Nrd1–Nab3–Sen1) interacts via Nrd1 with the CTD phosphorylated on Ser5, which is present at the 3′ ends of short genes, such as snoRNAs and CUTs. Ssu72 and Ess1 are also required to dephosphorylate Ser5-P, although the exact mechanism of Nrd1c-dependent termination awaits further studies.