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.
Highlights
MRNA capping occurs co-transcriptionally by a series of three enzymatic reactions in which the 5Ј-triphosphate terminus of the pre-mRNA is cleaved to a diphosphate by RNA triphosphatase, capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA methyltransferase [1]
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, but instead bind independently to the phosphorylated carboxyl-terminal domain (CTD)
S. pombe and Candida albicans guanylyltransferases can function in S. cerevisiae with the endogenous Cet1 triphosphatase [21, 23], but the ability of heterologous triphosphatases to function with the S. cerevisiae guanylyltransferase is variable and correlates with the presence or absence of a conserved guanylyltransferase-binding domain on the surface of the heterologous RNA triphosphatase [11, 24]
Summary
MRNA capping occurs co-transcriptionally by a series of three enzymatic reactions in which the 5Ј-triphosphate terminus of the pre-mRNA is cleaved to a diphosphate by RNA triphosphatase, capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA (guanine-7) methyltransferase [1]. S. pombe Triphosphatase and Guanylyltransferase Interact with S. pombe CTD in Vivo—A two-hybrid screen of ϳ100,000 transformants for triphosphatase-interacting proteins using BD-Pct1 as bait yielded 16 Hisϩ isolates, of which 8 contained plasmids encoding AD fused in-frame to a COOH-terminal fragment of Rpb1, the largest subunit of S. pombe pol II [26].
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