Crystal structure of the human symplekin–Ssu72–CTD phosphopeptide complex

Abstract

Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3′-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs) 1–4, and also participates in transcription initiation and termination by RNA polymerase II (Pol II) 5,6. Symplekin mediates interactions among many different proteins in this machinery 1,2,7–9, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the N-terminal domain (residues 30–340) of human symplekin (Symp-N) in a ternary complex with the Pol II C-terminal domain (CTD) Ser5 phosphatase Ssu72 7,10–17 and a CTD Ser5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of anti-parallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-weight phosphotyrosine protein phosphatase 18,19, although Ssu72 has a unique active site landscape as well as extra structural features at the C-terminus that is important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, unexpectedly with the pSer5-Pro6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations 20,21. While the active site of Ssu72 is about 25 Å away from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but importantly only when coupled to transcription. As catalytically active Ssu72 overcomes this inhibition, our results demonstrate a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3′-end processing.

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

“…Structures of the N-terminal 271 amino acids of Symplekin reveal seven pairs of α-helices comprising a HEAT domain ( Fig. 1A; Kennedy et al 2009;Xiang et al 2010). This N-terminal HEAT domain in human Symplekin mediates proteinprotein interactions and interacts directly with Ssu72, a RNA polymerase II carboxy-terminal domain (RNAPII CTD) Ser 5 phosphatase that is required for mRNA 3 ′ end processing in yeast (He et al 2003;Krishnamurthy et al 2004;Xiang et al 2010).…”

“…1A; Kennedy et al 2009;Xiang et al 2010). This N-terminal HEAT domain in human Symplekin mediates proteinprotein interactions and interacts directly with Ssu72, a RNA polymerase II carboxy-terminal domain (RNAPII CTD) Ser 5 phosphatase that is required for mRNA 3 ′ end processing in yeast (He et al 2003;Krishnamurthy et al 2004;Xiang et al 2010). The C-terminal 85 amino acids of the Symplekin yeast homolog, Pta1, interact with Ysh1 (the yeast CPSF73 homolog) in a directed yeast-two hybrid assay and full-length Pta1 interacts with yeast CPSF100 in an in vitro pull-down assay (Ghazy et al 2009).…”

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

“…Several structures have been published for Drosophila or human Ssu72 bound to CTD peptides of different phosphorylation states, including Drosophila Ssu72, Drosophila Ssu72-Symplekin and human Ssu72-Symplekin, bound to pSer5 CTD (PDB codes: 3P9Y 16 , 4IMJ 33 and 3O2Q 34 , respectively), and Drosophila Ssu72-symplekin bound to a CTD peptide with Thr4/Ser5 doubly phosphorylated (PDB code: 4IMI 33 ). The superimposition of these structures reveals that all known phosphoryl CTD peptides adopt a tight turn facilitated by cis-proline upon binding to Ssu72-symplekin (Fig.…”

RNA Polymerase II Phosphorylation and Gene Expression Regulation

“…Afterward, it was demonstrated that Ssu72 is part of the cleavage and polyadenylation factor (CPF) with a role at the 3'-end of genes [166,175]. In fact, Ssu72 is crucial for transcription-coupled 3'-end processing and termination of proteincoding genes [175,[186][187]. Later, Ssu72 was characterized as a Ser5P phosphatase [79] and a potential tyrosine phosphatase [188] and, most recently, it has been demonstrated that Ssu72 is also a Ser7 phosphatase [50,69].…”

“…As in yeast, mammalian Ssu72 associates with TFIIB and the yeast cleavage/polyadenylation factor Pta1, and exhibits intrinsic phosphatase activity [176]. The crystal complex structure that is formed by human symplekin (Pta1 in yeast), hSsu72 and a CTD phosphopeptide has been elucidated, and hSsu72 was demonstrated to have a function in coupling transcription to pre-mRNA 3'-end processing [187].…”