The Length, Phosphorylation State, and Primary Structure of the RNA Polymerase II Carboxyl-terminal Domain Dictate Interactions with mRNA Capping Enzymes

Department of Microbiology and Immunology, Cornell University, Итак, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2001; 276(30):28075-82. DOI: 10.1074/jbc.M102170200
Source: PubMed


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.

    • "Ser5 is the only phospho-acceptor amino acid in the Pol2 CTD that is strictly essential for growth of fission yeast (Schwer and Shuman 2011). Replacing all of the Ser5 residues in the consensus heptads with alanine is lethal because the Ser5-PO 4 mark is needed for recruitment of the fission yeast mRNA capping enzymes RNA triphosphatase and RNA guanylyltransferase to the Pol2 elongation complex (Pei et al. 2001; Doamekpor et al. 2014). The lethality of rpb1-CTD- S5A can be rescued by covalently fusing mammalian capping enzyme (MCE, a bifunctional RNA triphosphatase-guanylyltransferase ) to the mutant Rpb1-S5A polypeptide (Schwer and Shuman 2011). "
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    ABSTRACT: Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS(2)PTSPS repeat. The SpFcp1 catalytic domain (aa 140-580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1-139) and its putative metal-binding motif C(99)H(101)Cys(109)C(112) are essential; (iii) the carboxy-terminal domain (aa 581-723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330-393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T(1)PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg(2+) and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction.
    No preview · Article · May 2015 · RNA
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    • "The RNA guanine-7-methyltransferase (Abd1p) is recruited to the transcription initiation site via an interaction with phosphorylated RNA pol II CTD [12,95,173]. (b) In S. pombe, the RNA triphosphatase (Pct1p) and the RNA guanylyltransferase (Pce1p) independently interact with phosphorylated RNA pol II CTD [171,174]. Pct1p interacts with DSIF (DRB sensitivity-inducing factor) (Spt4/Spt5) and P-TEFb (Cdk9/Cyclin T1) [175,176]. Pce1p interacts with DSIF [176,177]. "
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    ABSTRACT: The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events.
    Full-text · Article · Jan 2014 · Biochemical Journal
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    • "Similarly, fission yeast (Schizosaccharomyces pombe) encodes separate triphosphatase (Pct1p) and guanylyltransferase (Pce1p). However , Pct1p and Pce1p do not interact with each other in S. pombe (Shuman et al. 1994; Pei et al. 2001a,b), while Ceg1p binds to a dimer of Cet1p to form a heteromeric stable capping enzyme complex in S. cerevisae (Shibagaki et al. 1992; Tsukamoto et al. 1997; Lehman et al. 1999; Fabrega et al. 2003). Although the capping enzymes are differentially organized among eukaryotes, their functions are evolutionarily conserved . "
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    ABSTRACT: Yeast mRNA 5'-triphosphatase, Cet1p, recognizes phosphorylated-RNA polymerase II as a component of capping machinery via Ceg1p for co-transcriptional formation of mRNA cap-structure that recruits cap-binding complex (CBC) and protects mRNA from exonucleases. Here, we show that the accumulation of RNA polymerase II at the promoter proximal site of ADH1 is significantly enhanced in the absence of Cet1p. Similar results are also found at other genes. Cet1p is recruited to the 5' end of the coding sequence, and its absence impairs mRNA-capping, and hence CBC recruitment. However, such an impaired recruitment of CBC does not enhance promoter proximal accumulation of RNA polymerase II. Thus, Cet1p specifically lowers the accumulation of RNA polymerase II at the promoter proximal site independently of mRNA cap-structure or CBC. Further, we show that Cet1's N-terminal domain which is not involved in mRNA-capping decreases promoter proximal accumulation of RNA polymerase II. An accumulation of RNA polymerase II at the promoter proximal site in the absence of Cet1's N-terminal domain is correlated with reduced transcription. Collectively, our results demonstrate a novel role of Cet1p in regulation of promoter proximal accumulation of RNA polymerase II independently of mRNA capping activity, and hence transcription in vivo.
    Preview · Article · Oct 2013 · Genetics
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