Interactions between Fission Yeast mRNA Capping Enzymes and Elongation Factor Spt5

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. mRNA capping occurs cotranscriptionally 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, then capped with GMP by RNA guanylyltransferase, and methylated at the N7 position of guanine by RNA (guanine-7) methyltransferase (1). Targeting of cap formation to transcripts made by RNA polymerase II (Pol-II)

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

“…Co-transcriptional recruitment of capping enzymes may be coordinated with promoterproximal pausing 15-17, 19, a common feature of pol II transcription of mammalian, but not yeast, genes 20 , 21. In support of this idea, pol II pauses at the 5′ end of the DHFR gene, and HCE localizes in the same region 22 .…”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…1D). The Spt4 binding domain of S. pombe Spt5 had previously been localized by twohybrid assays to the segment from amino acids 165-400 that includes two of the tryptic sites, Arg217 and Lys381 (Pei and Shuman 2002). Coexpression in bacteria of HisSpt4 and Spt5-(218-800) resulted in a stable complex of the two polypeptides after Ni-affinity chromatography (data not shown), suggesting that the entire polypeptide segment preceding the tryptic site at Arg217 is not strictly necessary for interaction with Spt4.…”

“…Based on our initial two-hybrid data showing that both the N-terminal 164-amino acid segment and the C-terminal 190-amino acid nonamer array of S. pombe Spt5 are dispensable for binding to Spt4 (Pei and Shuman 2002), we sought to physically characterize the complex of Spt4 with Spt5-(165-800). This was accomplished by coexpressing His-tagged Spt4 with untagged Spt5-(165-800) in bacteria and then purifying His-Spt4 by Ni-agarose chromatography (Fig.…”

“…The capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II McCracken et al 1997;Yue et al 1997;Ho and Shuman 1999;Pei et al 2001;Fabrega et al 2003). Capping enzymes also bind to the Pol II elongation factor Spt5 (Wen and Shatkin 1999;Pei and Shuman 2002), which, in conjunction with its binding partner Spt4, elicits an elongation arrest at promoter-proximal sites (Wada et al 1998a). The Spt5/Spt4-induced elongation arrest in metazoans is alleviated by the Cdk9 protein kinase, a subunit of positive transcription elongation factor b (P-TEFb) (Wada et al 1998b).…”

“…Spt5 is a large polypeptide (z1000-1200 amino acids) composed of multiple domain modules, including an acidic N-terminal domain, central NusG-like NGN (NusG N-terminal homology), and KOW domains (Hartzog et al 1998;Wade et al 1998a;Ponting 2002), and a CTD targeted for threonine phosphorylation Yamada et al 2006). Spt5 is essential for cell growth in budding and fission yeast (Swanson et al 1991;Pei and Shuman 2002). Saccharomyces cerevisiae Spt5 participates in a vast network of genetic and physical interactions with the Pol II elongation complex and mRNA processing systems (Lindstrom et al 2003; see also Saccharomyces Genome Database, www.yeastgenome.org).…”