The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes
ABSTRACT The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivo interactions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding of Schizosaccharomyces pombe guanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO4, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD.
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ABSTRACT: The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats. Dynamic remodeling of the CTD, especially its serine phosphorylation pattern, conveys informational cues about the transcription apparatus to a large ensemble of CTD-binding proteins. Our genetic dissection of fission yeast CTD function provides insights to the "CTD code." Two concepts stand out. First, the Ser2 requirement for transcription during sexual differentiation is bypassed by subtracting Ser7, signifying that imbalance in the phosphorylation array, not absence of a phospho-CTD cue, underlies a CTD-associated pathology. Second, the essentiality of Ser5 for vegetative growth is circumvented by covalently tethering mRNA capping enzymes to the CTD, thus proving that capping enzyme recruitment is a chief function of the Ser5-PO(4) mark. This illustrates that a key "letter" in the CTD code can be neutralized by delivering its essential cognate receptor to the transcription complex via an alternative route.Molecular cell 06/2011; 43(2):311-8. DOI:10.1016/j.molcel.2011.05.024 · 14.46 Impact Factor
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ABSTRACT: Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular capping enzymes is required for efficient formation of the 5' mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian capping enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended β-like conformation that docks Tyr1 and Ser5-PO(4) onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function in vivo. The location and composition of the CTD binding site on mammalian capping enzyme is distinct from that of a yeast capping enzyme that recognizes the same CTD primary structure. Thus, capping enzymes from different taxa have evolved different strategies to read the CTD code.Molecular cell 06/2011; 43(2):299-310. DOI:10.1016/j.molcel.2011.06.001 · 14.46 Impact Factor
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ABSTRACT: Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon-exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense-mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap-binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap-binding protein eIF4E. These and other data indicate that NMD targets CBP80-bound mRNA during a 'pioneer' round of translation, but whether nuclear eIF4E also binds nascent but dead-end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron-containing RNA and the C-terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80-bound but not eIF4E-bound mRNA. Each of these proteins is also detected on CBP80-bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.The EMBO Journal 08/2002; 21(13):3536-45. DOI:10.1093/emboj/cdf345 · 10.75 Impact Factor