RNA polymerase II conducts a symphony of pre-mRNA processing activities

Howe, Kenneth J.

doi:10.1016/s0167-4781(02)00460-8

Cited by 84 publications

(85 citation statements)

References 188 publications

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“…It is possible that after polyadenylation the polymerase was released from the template or it is possible that termination does not occur under the conditions used here, which stall the polymerase by removal of NTPs. The exact timing of cleavage, polyadenylation, and termination in vivo is still unclear (45). Recent experiments indicate that extended communication with the AAUAAA signal is needed (9).…”

Section: Discussionmentioning

confidence: 99%

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Adamson

Shutt

Price

2005

Journal of Biological Chemistry

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Cleavage and polyadenylation define the 3 ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5 portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5 capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.Processing of eukaryotic mRNA starts during transcription and is influenced by the RNA polymerase II elongation complex (1, 2). Capping, polyadenylation, and splicing have been seen to occur on nascent transcripts in vitro, and a variety of in vivo and in vitro approaches have strongly implicated the carboxyl-terminal domain (CTD) 2 of the large subunit of RNA polymerase II in connecting transcription with these events (3-6). Whereas many of the factors required for mRNA processing have been identified and characterized, much less is known about how the processing and transcription machinery functionally influence each other.3Ј End formation is linked to transcription. Although RNA polymerase II is capable of transcribing hundreds of kilobase pairs in a completely processive manner, after transcribing a functional polyadenylation signal the polymerase usually terminates within 1 kb (3, 7) in a process that requires the CTD (8, 9). Factors required for polyadenylation have been found to associate with isolated transcription complexes (10) and the CTD has been implicated in bringing in the factors (11, 12). There is some controversy about when polyadenylation factors associate with the transcription complex. Polyadenylation factors have been found to associate with promoter binding factors (13), and yeast chromatin immunoprecipitation experiments in one study have localized the factors throughout the gene (11), but in another only at the 3Ј end of genes after the passage of a functional polyadenylation signal (14). Recent studies in yeast (15), Drosophila (16), and Xenopus (17) have implicated the CTD kinase positive transcription elongation factor b (P-TEFb) in promoting efficient polyadenylation. Drosophila histone mRNA 3Ј end formation, involving a completely different set of factors, was found not to be stimulated by having the RNA in an elongation complex (18). However, a strong transcriptional pause was found a...

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Section: Discussionmentioning

confidence: 99%

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Adamson

Shutt

Price

2005

Journal of Biological Chemistry

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“…1 transcription elongation and to mRNA processing, based on either physical association with pol II, cotranscriptional association with transcribed DNA, or association with the nascent RNA (1)(2)(3)(4). In Saccharomyces cerevisiae, these include factors involved in mRNA capping, splicing, termination, and export.…”

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confidence: 99%

Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus

Kaplan

Holland

Winston

2005

Journal of Biological Chemistry

112

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Spt6 is a conserved transcription factor that associates with RNA polymerase II (pol II) during elongation. Spt6 is essential for viability in Saccharomyces cerevisiae and regulates chromatin structure during pol II transcription. Here we present evidence that mutations that impair Spt6, a second elongation factor, Spt4, and pol II can affect 3-end formation at GAL10. Additional analysis suggests that Spt6 is required for cotranscriptional association of the factor Ctr9, a member of the Paf1 complex, with GAL10 and GAL7, and that Ctr9 association with chromatin 3 of GAL10 is regulated by the GAL10 polyadenylation signal. Overall, these results provide new evidence for a connection between the transcription elongation factor Spt6 and 3-end formation in vivo.Many factors are believed to contribute to pol II 1 transcription elongation and to mRNA processing, based on either physical association with pol II, cotranscriptional association with transcribed DNA, or association with the nascent RNA (1-4). In Saccharomyces cerevisiae, these include factors involved in mRNA capping, splicing, termination, and export. They also include the highly conserved and mostly essential elongation factors, the Spt4/Spt5 complex, Spt6, and the Spt16/Pob3 complex. Additionally, the Paf1 complex (comprising Paf1, Ctr9, Rtf1, Leo1, and Cdc73), Isw1, Iws1, the Set1 complex, Set2, and Chd1 have also been implicated as part of the pol II elongation complex (5-16).Previous work has shown that Spt6 is broadly utilized in pol II transcription (17,18), and data from several laboratories implicates Spt6 as a pol II elongation factor (16, 18 -20). Spt6 has been shown to interact with histones and is involved in the organization of chromatin structure over transcribed regions (21-23). Spt6 has also been implicated in RNA processing, because Drosophila Spt6 is associated with the nuclear exosome (24). Additional roles for Spt6 in the transcription cycle probably also exist.The Paf1 complex is a multisubunit complex that associates with pol II and localizes to transcribed regions (14,16,(25)(26)(27)(28)(29). Although all of the roles of the Paf1 complex remain to be elucidated, recently, some complex members have been shown to be required for cotranscriptional ubiquitylation of histone H2B at position 123 (H2B Lys-123) and methylation of histone H3 lysines at positions 4 (Lys 4 ), 36 (Lys 36 ), and 79 (Lys 79 ) (30 -34). Thus, the Paf1 complex appears to coordinate histone modifications with transcription. Mutations in genes encoding Paf1 complex members also exhibit a wide spectrum of genetic interactions with mutations in elongation factor genes such as SPT4, SPT5, SPT16, and POB3 (14,35,36).As pol II goes through the cycle of transcription initiation, elongation, and termination, it is presented with different tasks. Many of these tasks are coordinated through the phosphorylation of the largest subunit of pol II on its conserved C-terminal domain, which in yeast consists of 26 repeats of consensus Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 ...

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“…The principal function of this C-terminal repeat domain (CTD), which comprises multiple repeats of a consensus heptamer Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is to serve as a binding scaffold for various nuclear factors (reviewed in refs. [1][2][3]. The CTD of preinitiating RNA polymerase II is mostly unphosphorylated, whereas after initiation and during elongation it is hyperphosphorylated, principally on Ser-5 and Ser-2 residues of the repeats (4-6); we refer to this form as the phospho-CTD (PCTD).…”

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confidence: 99%

Solution structure of the Set2–Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1

Phatnani

Guan

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

118

110

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The phosphorylation state of the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II changes as polymerase transcribes a gene, and the distinct forms of the phospho-CTD (PCTD) recruit different nuclear factors to elongating polymerase. The Set2 histone methyltransferase from yeast was recently shown to bind the PCTD of elongating RNA polymerase II by means of a novel domain termed the Set2-Rpb1 interacting (SRI) domain. Here, we report the solution structure of the SRI domain in human Set2 (hSRI domain), which adopts a left-turned three-helix bundle distinctly different from other structurally characterized PCTDinteracting domains. NMR titration experiments mapped the binding surface of the hSRI domain to helices 1 and 2, and Biacore binding studies showed that the domain binds preferably to [Ser-2 ؉ Ser-5]-phosphorylated CTD peptides containing two or more heptad repeats. Point-mutagenesis studies identified five residues critical for PCTD binding. In view of the differential effects of these point mutations on binding to different CTD phosphopeptides, we propose a model for the hSRI domain interaction with the PCTD. RNA polymerase II carries an intrinsically unstructured, flexible domain at the C terminus of its largest subunit. The principal function of this C-terminal repeat domain (CTD), which comprises multiple repeats of a consensus heptamer Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is to serve as a binding scaffold for various nuclear factors (reviewed in refs. 1-3). The CTD of preinitiating RNA polymerase II is mostly unphosphorylated, whereas after initiation and during elongation it is hyperphosphorylated, principally on Ser-5 and Ser-2 residues of the repeats (4-6); we refer to this form as the phospho-CTD (PCTD). Attendant with changes in patterns of CTD phosphorylation, the ensemble of CTD-bound proteins changes as RNA polymerase II progresses through the transcription cycle (5). Although knowledge of the number and types of PCTD-associating proteins (PCAPs) has expanded rapidly (7, 8), information about the molecular nature of PCAP-PCTD interactions remains quite limited; detailed binding properties and͞or 3D structures are known for only a few PCTD-interacting domains (PCIDs) (9-14).Describing in molecular detail the interactions between the PCTD and its binding partners is an important step in advancing our understanding of the PCTD and its manifold functions. One recently identified binding partner of the PCTD is the Saccharomyces cerevisiae Set2 (yeast Set2; ySet2), a histone methyltransferase that modifies K36 of histone H3 in nucleosomes of transcribed genes (15)(16)(17)(18)(19). The identification of ySet2 as a PCAP together with studies of transcription in set2 mutant strains suggests a role for the PCTD in chromatin structure modulation during elongation (20). Deletion studies of ySet2 mapped a novel PCID, termed the Set2-Rpb1 interacting (SRI) domain by Kizer et al. (20), to the C-terminal segment of ySet2. The yeast SRI domain binds preferentially to PCTD peptides with both S...

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RNA polymerase II conducts a symphony of pre-mRNA processing activities

Cited by 84 publications

References 188 publications

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus

Solution structure of the Set2–Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1

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