Fission yeast RNA triphosphatase reads an Spt5 CTD code

Abstract

mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an "Spt5 CTD code" in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on-off switch that is read by diverse CTD receptors, each in its own distinctive manner.

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

“…The Thr1-PO 4 mark (installed by Cdk9) is thought to be important for the positive transcription elongation activity of fission yeast Spt5, insofar as changing all Spt5 CTD Thr1 positions to alanine sensitized S. pombe cells to growth inhibition by 5-azauracil, whereas changing Thr1 to a phosphomimetic glutamate did not (Schneider et al 2010). On the other hand, Thr1 phosphorylation antagonizes the interaction of the Spt5 CTD with the S. pombe mRNA capping enzymes (Doamekpor et al 2014(Doamekpor et al , 2015. Genetic evidence in fission yeast indicates that the unphosphorylated Spt5 CTD and the Ser5-phosphorylated Pol2 CTD collaborate in vivo to recruit the capping apparatus to the Pol2 elongation complex.…”

“…Unlike the capping enzyme·Pol2-CTD interactions, which stringently depend on the Ser5-PO 4 mark, binding of fission yeast triphosphatase and guanylyltransferase to the Spt5 CTD is independent of Thr1 phosphorylation (Pei et al 2001;Pei and Shuman 2002). Indeed, their binding to the Spt5 CTD is antagonized by Thr1 phosphorylation (Doamekpor et al 2014(Doamekpor et al , 2015. The phosphatase(s) responsible for removing the Thr1-PO 4 mark from the Spt5 CTD have not been defined.…”

THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing

“…In mammals, CTD phosphorylation has a 4-fold effect on capping of soluble RNA (Ho and Shuman, 1999) or nascent Pol II transcripts (Mandal et al, 2004; Moteki and Price, 2002). Capping enzymes have also been shown to interact with the Spt5 subunit of DSIF in yeast (Doamekpor et al, 2014; Doamekpor et al, 2015; Pei and Shuman, 2002; Schneider et al, 2010) and human DSIF causes a 2 to 5-fold stimulation of co-transcriptional capping (Mandal et al, 2004; Wen and Shatkin, 1999). Additionally, wild type or capping-defective HCE relieved the negative influence of NELF during transcription in vitro suggesting a competition of HCE and pausing factors for Pol II (Mandal et al, 2004).…”

Spt5 Plays Vital Roles in the Control of Sense and Antisense Transcription Elongation

“…1A), a sequence of tandem repeats of amino acids that are phosphorylated in a manner similar to the CTD of RNAPII (Hartzog and Fu, 2013). The Spt5 CTR in its unphosphorylated state aids in recruiting the mRNA capping enzyme (Doamekpor et al, 2014; Doamekpor et al, 2015; Schneider et al, 2010; Wen and Shatkin, 1999). In contrast, the phosphorylated Spt5 CTR is required for recruitment of the Paf1 complex, which plays an important role in elongation by RNAPII (Liu et al, 2009; Mbogning et al, 2013; Wier et al, 2013; Zhou et al, 2009).…”