Reference: Pei Y and Shuman S (2002) Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5. J Biol Chem 277(22):19639-48

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Abstract

Elongating RNA polymerase II is targeted by macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA. Although recruitment of the capping enzymes to the transcription complex is dependent on phosphorylation of the C-terminal domain of the Rpb1 subunit of polymerase II (Pol-II), there may be additional levels of control that coordinate capping with elongation. Here we show that the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the fission yeast capping apparatus bind independently to the elongation factor Spt5. The C-terminal domain of the 990-amino acid Schizosaccharomyces pombe Spt5 protein, composed of repeats of a nonapeptide motif (consensus sequence TPAWNSGSK), is necessary and sufficient for binding to the capping enzymes in vivo (in a two-hybrid assay) and in vitro. As few as four nonamer repeats suffice for Spt5 binding to Pct1 in vitro, whereas six repeats are required for Spt5 binding to Pce1. A 116-amino acid fragment of the guanylyltransferase Pce1 suffices for binding to the Spt5 C-terminal domain (CTD) but not for binding to the Pol-II CTD. Pct1 and Pce1 can bind simultaneously to the Spt5 CTD in vitro. We find that Spt5 is essential for viability of S. pombe and that it interacts in vivo with S. pombe Spt4 via a central domain distinct from the Spt5 CTD. We suggest that Spt5-induced arrest of elongation at promoter proximal positions ensures a temporal window for recruitment of the capping enzymes.

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Journal Article
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Pei Y, Shuman S
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