Ssu72 Protein Mediates Both Poly(A)-Coupled and Poly(A)-Independent Termination of RNA Polymerase II Transcription

Abstract

Termination of transcription by RNA polymerase II (Pol II) is a poorly understood yet essential step in eukaryotic gene expression. Termination of pre-mRNA synthesis is coupled to recognition of RNA signals that direct cleavage and polyadenylation of the nascent transcript. Termination of nonpolyadenylated transcripts made by Pol II in the yeast Saccharomyces cerevisiae, including the small nuclear and small nucleolar RNAs, requires distinct RNA elements recognized by the Nrd1 protein and other factors. We have used genetic selection to characterize the terminator of the SNR13 snoRNA gene, revealing a bipartite structure consisting of an upstream element closely matching a Nrd1-binding sequence and a downstream element similar to a cleavage/polyadenylation signal. Genome-wide selection for factors influencing recogniton of the SNR13 terminator yielded mutations in the gene coding for the essential Pol II-binding protein Ssu72. Ssu72 has recently been found to associate with the pre-mRNA cleavage/polyadenylation machinery, and we find that an ssu72 mutation that disrupts Nrd1-dependent termination also results in deficient poly(A)-dependent termination. These findings extend the parallels between the two termination pathways and suggest that they share a common mechanism to signal Pol II termination.A productive cycle of transcription requires the recruitment of RNA polymerase to a promoter, initiation of RNA synthesis, elongation of the nascent RNA chain, and termination of transcription to release the completed RNA and the polymerase from the DNA template (28). Accurate and efficient termination is critical to prevent interference between adjacent transcription units and to allow recycling of RNA polymerase. Distinct mechanisms of termination characterize the three eukaryotic nuclear RNA polymerases (Pols). Pol III, which synthesizes small noncoding RNAs including tRNAs, 5S rRNA, and U6 snRNA, has an intrinsic ability to terminate transcription upon incorporation of 4 to 6 contiguous U residues (1, 10). Pol I transcribes rRNA genes and, like Pol III, terminates within a U-rich region. However, Pol I termination is also dependent on an elongational pause caused by Reb1 protein bound to DNA downstream of the termination site (40).Pol II synthesizes transcripts with a great diversity of sizes and functions, and the control of termination by Pol II is more complex and less well understood than termination by Pol I or Pol III. The mature 3Ј ends of most protein-coding Pol II transcripts (mRNAs) are generated by endonucleolytic cleavage followed by addition of a poly(A) tail to the newly generated 3Ј end (58). This 3Ј processing is carried out by a complex machinery that associates with Pol II during elongation (37) and engages processing signals in the nascent transcript even as the polymerase continues to transcribe beyond the processing site. Thus, the production of a mature mRNA 3Ј end does not require termination of transcription. Nevertheless, pre-mRNA 3Ј processing and termination of Pol II transcription are c...

Transcription termination by nuclear RNA polymerases

“…This provides a signal for binding many factors involved in 39-end formation, which in turn renders RNAPII competent for termination (Whitelaw and Proudfoot 1986;Logan et al 1987;Connelly and Manley 1988;Birse et al 1997;. In yeast, mutations and conditional depletion of factors involved in pre-mRNA cleavage and polyadenylation, including homologs of CstF-64 (Rna15), CstF-77 (Rna14), Pcf11, CPSF160 (Yhh1), CPSF-73 (Ysh1), and Ssu72, result in read-through at the 39-end of protein-coding genes (Birse et al 1998;Dichtl et al 2002;Steinmetz and Brow 2003;Garas et al 2008). Accordingly, ChIP assays suggest that 39-end processing factors, including Pcf11, Rna14, and Rna15, become associated with the RNAPII EC at the poly(A) site, in a Ser2 phosphorylated CTD-dependant manner (Ahn et al 2004;Kim et al 2004a;Luo et al 2006).…”

“…2; Fatica et al 2000;Morlando et al 2002;Steinmetz and Brow 2003;Steinmetz et al 2006a). Both terminators are required for efficient termination.…”

“…Additionally, mutations in a subcomplex of the cleavage/ polyadenylation machinery, the APT complex (associated with Pta1 [homolog of symplekin]), lead to read-through transcription of certain sn/snoRNA genes (Dheur et al 2003;Ganem et al 2003;Nedea et al 2003;Steinmetz and Brow 2003;Kim et al 2006;Ghazy et al 2009). The APT factors implicated in sn/snoRNA termination include Pti1, a second yeast homolog of CstF-64 that also binds Rna14; Ref2, which interacts with the snoRNPspecific protein Nop1 (Morlando et al 2004); the CTD phosphatase Ssu72 (Ganem et al 2003;Steinmetz and Brow 2003;Kim et al 2006); and Swd2, which is also a component of the Set1 complex (Cheng et al 2004;Dichtl et al 2004). Overexpression of Pti1 inhibits polyadenylation without affecting cleavage, suggesting that Pti1 may function in uncoupling cleavage and polyadenylation during snoRNA 39-end formation (Dheur et al 2003).…”

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…In contrast to the four kinases, there are two known CTD phosphatases in yeast, . Biochemical as well as genetic evidence indicates that both proteins may function as CTD phosphatases in the context of transcription (16,19,20,(25)(26)(27)(28)(29)(30)(31).It is the interplay between these CTD kinases and phosphatases that influences which factors associate with transcribing polymerase and when during the transcription cycle such associations occur. As mentioned, the recruitment of capping enzyme early in the transcription cycle is believed to be one consequence of CTD phosphorylation on Ser5 by Kin28 (6,7).…”

RNA Polymerase II Phosphorylation and Gene Expression Regulation

“…In fact, Ssu72 was originally identified as functionally interacting with the general transcription factor TFIIB [184][185]. Afterward, it was demonstrated that Ssu72 is part of the cleavage and polyadenylation factor (CPF) with a role at the 3'-end of genes [166,175]. In fact, Ssu72 is crucial for transcription-coupled 3'-end processing and termination of proteincoding genes [175,[186][187].…”