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

Abstract

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 ...

Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae

“…3B), and found that Rpb1 is distributed similarly to Spt2. Overall, the distribution of Spt2 resembles the pattern previously determined for the elongation factors Spt4, Spt6, and Spt16 (17,19). It is interesting to note that this pattern is distinct from that of other elongation factors, including the PAF and Tho/Trex complexes, which are not associated with the 3ЈUTRs of PMA1 and other genes (17,19).…”

Potential role of the histone chaperone, CAF-1, in transcription

“…Their recruitment profile was further analyzed by normalization with pol II (Fig. 1B, bottom panel), and resembled the association pattern of the elongation factors, Spt2, Spt4, Spt5, Spt6, Spt16, and Elf1, all involved in chromatin dynamics, whose association persists over the 3'UTR (14,(16)(17)(18).…”

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

“…Spt6 also has a role in the re-assembly of nucleosomes in promoter regions, and is required for repression of transcription reinitiation. 10 Mutations in Spt6 influence mRNA 3′-end formation, 11 splicing, and mRNA export. 12 Spt6 is associated with the Pol II machinery, 6,7 , and its SH2 domain is apparently important for this.…”