Dynamic association of capping enzymes with transcribing RNA polymerase II

Abstract

The C-terminal heptad repeat domain (CTD) of RNA polymerase II (pol II) is proposed to target pre-mRNA processing enzymes to nascent pol II transcripts, but this idea has not been directly tested in vivo. In vitro, the yeast mRNA capping enzymes Ceg1 and Abd1 bind specifically to the phosphorylated CTD. Here we show that yeast capping enzymes cross-link in vivo to the 5 ends of transcribed genes and that this localization requires the CTD. Both the extent of CTD phosphorylation at Ser 5 of the heptad repeat and the binding of capping enzymes decreased as polymerase moved from the 5 to the 3 ends of the ACT1, ENO2, TEF1, GAL1, and GAL10 genes. Ceg1 is released early in elongation, but Abd1 can travel with transcribing pol II as far as the 3 end of a gene. The CTD kinase, Kin28, is required for binding, and the CTD phosphatase, Fcp1, is required for dissociation of capping enzymes from the elongation complex. CTD phosphorylation and dephosphorylation therefore control the association of capping enzymes with pol II as it transcribes a gene. The conserved CTD of the pol II large subunit has dual functions in controlling transcriptional responses (Scafe et al. 1990;Gerber et al. 1995) and in coordinating premRNA synthesis with processing. It has been suggested that the CTD serves as a landing pad for processing factors and thereby targets them specifically to transcripts made by pol II and not by other RNA polymerases (Yuryev et

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

“…Remarkably, mammalian capping enzyme binds synthetic CTD peptides containing phosphoserine at either position 2 or 5, but only Ser-5 phosphorylation stimulates guanylyltransferase activity (Ho and Shuman, 1999). This observation correlates well with recent observations in yeast suggesting that Ser-5 phosphorylation by the CDK7 kinase component of TFIIH during the transition from transcription inititation to elongation (Dahmus, 1996;Yamamoto et al, 2001) might serve, at least in part, to target and activate the capping machinery (Komarnitsky et al, 2000;Schroeder et al, 2000).…”

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

“…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). Consistent with this, capping enzyme and Kin28 interact genetically (6).…”

“…These different CTD phosphorylation patterns serve as a guide for the timely recruitment to transcribing Pol II of factors that play a role in transcript biogenesis. For example, Ser5 phosphorylation is important for recruiting capping enzyme early in the transcription cycle (6,7), whereas Ser2 phosphorylation plays a role in recruiting Pcf11, a factor involved in 3′-end formation (8). Modulating the extent and pattern of CTD phosphorylation can thus be an effective way of regulating the CTD's affinity for numerous protein factors, as it can potentially generate a profuse array of different phosphoepitopes (9).…”

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

“…The recruitment patterns we observed for human capping enzymes differ from their yeast counterparts. Yeast Ceg1 and Abd1 localize at 5′ ends, and Abd1 is also found within genes, but neither has been detected in 3′ flanking regions 7,43 .…”