A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

Abstract

RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.In eukaryotic cells, RNA polymerase II (RNA Pol II) 5 and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1-4), mRNA export, and chromatin reconfiguration (5-7). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8 -10) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.The formation of RNA 5Ј cap structure, m 7 GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ϳ25 nucleotides (12-15). The RNA cap is formed in three enzymatic steps (16, 17); (i) removal of the 5Ј ␥-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5Ј diphosphate by the guanylyltransferase; (iii) methylation of the 5Ј guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most org...

Correct Assembly of RNA Polymerase II Depends on the Foot Domain and Is Required for Multiple Steps of Transcription in Saccharomyces cerevisiae

“…In S. cerevisiae, Ser5 phosphorylation occurs first in the CTD in coordination with the recruitment of Ceg1 (47), which binds the foot of RNA pol II in S. cerevisiae (14). In agreement, we demonstrated that the foot is in contact with proteins that participate in transcription initiation and/or early elongation (13) and that interact genetically with CEG1.…”

“…Suh et al have reported previously that mutating the foot domain significantly reduced the amount of CE associated with RNA pol II in S. cerevisiae (14). Thus, if the impairment of the Ceg1 interaction with RNA pol II is responsible for the slowgrowth phenotypes of the rpo21-4 and rpb1-84 mutants, overexpression of CEG1 might compensate for these phenotypes.…”

“…We and others have shown that the foot of RNA pol II associates with proteins involved in transcription initiation or early elongation (13,14). However, the role of this region in transcription remains to be elucidated.…”

“…It should be noted that we and others have identified interactions between the foot and proteins involved in transcription initiation and/or early elongation, such as the regulators Mvp1 and Spo14 and the RNAcapping enzyme (CE) in Saccharomyces cerevisiae (13,14). Moreover, evidence from manipulating the foot indicates that this domain contributes to specificity in the interaction of the CE with RNA pol II, as opposed to RNA polymerases I and III (14). In addition, some authors have proposed that the rpo21-4 mutation in the foot of RNA pol II of S. cerevisiae affects the assembly or the integrity of RNA pol II (15)(16)(17).…”

“…This domain, conserved among RNA pol II enzymes from different species, is one of the two regions in the largest subunit that differ the most in sequence among RNA polymerases I, II, and III (10,13,14). It should be noted that we and others have identified interactions between the foot and proteins involved in transcription initiation and/or early elongation, such as the regulators Mvp1 and Spo14 and the RNAcapping enzyme (CE) in Saccharomyces cerevisiae (13,14). Moreover, evidence from manipulating the foot indicates that this domain contributes to specificity in the interaction of the CE with RNA pol II, as opposed to RNA polymerases I and III (14).…”