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.
The 5′→ 3′ exoribonucleases (XRNs) have important functions in transcription, RNA metabolism, and RNA interference. The recent structure of Rat1 (Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain, defining the active site of the enzyme. Xrn1 has a 510-residue segment following the conserved regions that is required for activity but is absent in Rat1. We report here the crystal structures at 2.9 Å resolution of Kluyveromyces lactis Xrn1 (residues 1–1245, E178Q mutant), alone and in complex with a Mn2+ ion in the active site. The 510-residue segment contains four domains (D1–D4), located far from the active site. Our mutagenesis and biochemical studies demonstrate that their functional importance is due to their stabilization of the conformation of the N-terminal segment of Xrn1. These domains may also constitute a platform for interacting with protein partners of Xrn1.
Processing of mRNA precursors (pre-mRNAs) by polyadenylation is an essential step in gene expression. Polyadenylation consists of two steps, cleavage and poly(A) synthesis, and requires multiple cis elements in the pre-mRNA and a megadalton protein complex bearing the two essential enzymatic activities. While genetic and biochemical studies remain the major approaches in characterizing these factors, structural biology has emerged during the past decade to help understand the molecular assembly and mechanistic details of the process. With structural information about more proteins and higher-order complexes becoming available, we are coming closer to obtaining a structural blueprint of the polyadenylation machinery that explains both how this complex functions and how it is regulated and connected to other cellular processes.
In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.
Ssu72, an RNA polymerase II C-terminal domain (CTD) phospho-Ser5 (pSer5) phosphatase, was recently reported to have pSer7 phosphatase activity as well. We report here the crystal structure of a ternary complex of the N-terminal domain of human symplekin, human Ssu72, and a 10-mer pSer7 CTD peptide. Surprisingly, the peptide is bound in the Ssu72 active site with its backbone running in the opposite direction compared with a pSer5 peptide. The pSer7 phosphatase activity of Ssu72 is~4000-fold lower than its pSer5 phosphatase activity toward a peptide substrate, consistent with the structural observations. Transcription of mRNA and noncoding RNA in eukaryotes is carried out by RNA polymerase II (Pol II), the activity of which is regulated in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit (Komarnitsky et al. 2000;Schroeder et al. 2000;Meinhart et al. 2005;Phatnani and Greenleaf 2006;Buratowski 2009;Kim et al. 2009Kim et al. , 2010Mayer et al. 2010;Tietjen et al. 2010). The CTD contains the consensus heptapeptide repeat Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (YSPTSPS), and phosphorylation of Ser5 and Ser2 has long been recognized for its importance in transcription and coupled RNA processing events. Phosphorylation of Ser7 is involved in snRNA transcription and 39 end processing Egloff et al. 2007), and phosphorylation of Thr4 has been linked to histone mRNA 39 end processing (Hsin et al. 2011). CTD kinases and phosphatases control the phosphorylation state of the CTD and thereby regulate Pol II activity. For example, Fcp1 preferentially dephosphorylates pSer2 over pSer5 (Hausmann and Shuman 2002;Hausmann et al. 2004;Ghosh et al. 2008), while Ssu72 is a pSer5 phosphatase (Krishnamurthy et al. 2004;Hausmann et al. 2005). Both are essential for viability in yeast.Recently, it was reported that Ssu72 also has pSer7 phosphatase activity (Bataille et al. 2012;Zhang et al. 2012). However, such an activity for Ssu72 is unexpected from a structural perspective. Ssu72 recognizes the cis configuration of the pSer5-Pro6 peptide bond as a pSer5 phosphatase (Xiang et al. 2010;Werner-Allen et al. 2011). In comparison, pSer7 is followed by Tyr1 in the next repeat of the CTD (designated Tyr19). If Ssu72 were to bind the pSer5 and pSer7 substrates in the same way, the pSer7-Tyr19 peptide bond would need to be in the cis configuration, which is much less favorable energetically. Moreover, the bulkier Tyr side chain would be placed in the binding site for the Pro6 residue in the pSer5 substrate, which would clash with the enzyme. Results and DiscussionTo understand the structural basis for how Ssu72 functions as a pSer7 phosphatase, we determined the crystal structure at 2.2 Å resolution of a ternary complex of a human symplekin N-terminal domain (NTD, residues 30-360), human Ssu72 (C12S mutant), and a 10-mer CTD peptide phosphorylated at Ser7 (Ser2-Pro3-Thr4-Ser5-Pro6-pSer7-Tyr19-Ser29-Pro39-Thr49, with the prime indicating the next repeat of the CTD) (Fig. 1A). Symplekin is a scaffold protein in t...
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