Cap Completion and C-Terminal Repeat Domain Kinase Recruitment Underlie the Initiation-Elongation Transition of RNA Polymerase II

Abstract

After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. The molecular basis for the initiation-elongation factor exchange during this transition remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to elucidate the initiation-elongation transition of Pol II in the budding yeast Saccharomyces cerevisiae. We show that the early Pol II elongation factor Spt5 contributes to stable recruitment of the mRNA capping enzymes Cet1, Ceg1, and Abd1. Genome-wide occupancy for Cet1 and Ceg1 is restricted to the transcription start site (TSS), whereas occupancy for Abd1 peaks at ϳ110 nucleotides downstream, and occupancy for the cap-binding complex (CBC) rises subsequently. Abd1 and CBC are important for recruitment of the kinases Ctk1 and Bur1, which promote elongation and capping enzyme release. These results suggest that cap completion stimulates productive Pol II elongation.T ranscription of protein-coding genes by RNA polymerase (Pol) II begins with assembly of an initiation complex at the promoter. As the RNA grows, initiation factors dissociate from Pol II, and elongation factors are recruited (1-3). The initiationelongation transition begins when the nascent RNA grows to about 12 nucleotides (nt), which releases initiation factor TFIIB (4, 5). Release of initiation factors frees the Pol II clamp domain, which binds the conserved elongation factor Spt5 (6, 7). Here, we refer to the exchange of initiation by elongation factors as the initiation-elongation transition. The transition starts with the dissociation of initiation factors and ends with completed recruitment of elongation factors.During initiation, the C-terminal repeat domain (CTD) of Pol II gets phosphorylated at the serine 5 (S5) and S7 residues (8-10). CTD S5 phosphorylation is important for recruitment of RNA 5= capping enzymes (11-13). Capping starts with removal of the terminal ␥-phosphate from the 5= triphosphate end of the nascent RNA, which is catalyzed by the RNA triphosphatase (14-16). The truncated RNA 5= end is then linked to an inverted guanylyl group by the guanylyltransferase. Finally, the cap methyltransferase methylates position N7 of the newly added terminal guanine. In Saccharomyces cerevisiae the three catalytic activities are encoded by Cet1, Ceg1, and Abd1, respectively. In metazoans the first two steps of the capping reaction are catalyzed by a single capping enzyme consisting of an N-terminal triphosphatase and a C-terminal guanylyltransferase domain. The resulting 7-methylguanosine (m7G) cap protects the transcript from degradation and promotes translation initiation of the mRNA (17, 18). The complete cap associates with the cap-binding complex (CBC) that functions in pre-mRNA splicing and mRNA export (19,20).We have previously obtained high-resolution genome-wide occupancy profiles for Pol II initiation and elongation factors by chromatin immunoprecipitation (ChIP) in S. cerevisiae. These studies showed that initiation factor occupancy peaked around 50...

A Novel Role for Cet1p mRNA 5′-Triphosphatase in Promoter Proximal Accumulation of RNA Polymerase II in Saccharomyces cerevisiase

“…CBC plays an important role in the initiation-elongation transition of RNA polymerase II by recruiting kinases such as Ctk1p and Bur1p (Lidschreiber et al 2013). Further, CBC has been shown to regulate transcriptional elongation via histone H3K36 methylation and histone H2B ubiquitylation (Hossain et al 2009(Hossain et al , 2013.…”

“…The cap structure recruits cap-binding complex (CBC) that plays crucial roles in maintaining the stability of mRNA and its splicing, export, and translation (Izaurralde et al 1994;Colot et al 1996;Izaurralde et al 1995;Flaherty et al 1997;Schwer et al 1998;Shatkin and Manley 2000;Shen et al 2000;Ishigaki et al 2001;McKendrick et al 2001;Maquat 2004;Gornemann et al 2005;Hosoda et al 2005;Svitkin et al 2005;Balatsos et al 2006;Nojima et al 2007;Wong et al 2007;Kuhn et al 2008;Laubinger et al 2008). Further, CBC has been recently implicated in controlling transcriptional initiation and elongation, histone H2B ubiquitylation, histone H3K36 (lysine 36) methylation, RNA interference, cell proliferation, and processing of primary microRNAs (Kim et al 2008;Gruber et al 2009;Hossain et al 2009Hossain et al , 2013Nielsen et al 2009;Sabin et al 2009;Lahudkar et al 2011;Lidschreiber et al 2013). Thus, the 59 end capping of mRNA has critical functions in eukaryotic gene expression.…”

RNA Polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation

“…Reducing Bur1 activity does not increase promoter proximal Pol II levels. 141 Instead, there is a reduction in both initiated and elongating Pol II. 119,141,142 As in budding yeast, the Bur1 homolog, Cdk9 is not required for elongation in fission yeast.…”

“…141 Instead, there is a reduction in both initiated and elongating Pol II. 119,141,142 As in budding yeast, the Bur1 homolog, Cdk9 is not required for elongation in fission yeast. 143 Thus, while Bur1/Cdk9 phosphorylate the CTD toward the 5′ end of genes, is not required for the transition of Pol II into productive elongation in yeast.…”

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

“…This latter hypothesis was explored by ChIP experiments using tagged Cbc2p subunit of the CBC complex. 31 However, the results did not allow us to draw a clear conclusion since no significant difference in the recruitment could be observed between the presence and the absence of Rho.…”