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...

The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 plays a noncatalytic role in CTD dephosphorylation.

Type 2 diabetes, characterized by chronic hyperglycemia caused by insulin resistance and β-cell dysfunction, has become a worldwide public health problem, 1 leading to considerable emphasis on the management of type 2 diabetes. Oral antidiabetic drugs are among the most widely prescribed of all medications for lowering of glucose levels. These include metformin, sulfonylureas, glinides, α-glucosidase inhibitors, and thiazolidinediones. 2 However, there is interindividual variability in the responses to these drugs, partly attributable to genetic factors implicated in drug absorption, distribution, metabolism, and target. 3 Variants in CYP2C8, SLCO1B1, KCNJ11, ABCC8, NOS1AP, and TCF7L2 were previously found to be associated with repaglinide efficacy, 4-8 whereas variants in CYP2C8, PPARG, UCP2, ADRB3, TNF-α, and ABCA1 were shown to be associated with rosiglitazone treatment outcome. [9][10][11][12][13] Recently, after genome-wide association studies in East Asians, 14,15 replicated in the population of mainland China, 16 KCNQ1 has been identified as a susceptibility gene for type 2 diabetes. However, whether KCNQ1 single-nucleotide polymorphisms (SNPs) have an impact on the therapeutic effects of oral antihyperglycemic drugs remains unknown. Consequently, we conducted this study aimed at evaluating the effects of these polymorphisms on the efficacy of repaglinide and rosiglitazone in newly diagnosed patients with type 2 diabetes. ResultsA total of 91 patients treated with repaglinide completed the 48-week follow-up. Of the 13 subjects who were withdrawn from the study, 4 had glycated hemoglobin (HbA 1c ) ≥8% at two consecutive time points, and 9 were lost to follow-up. Simultaneously, in the rosiglitazone cohort, 1 patient with elevated hepatic enzymes and 5 patients with inadequate control of blood glucose or HbA 1c were excluded, and 6 patients were lost to follow-up, leaving a sample of 93 patients who completed the whole study (see Supplementary Table S1 online).All the SNPs conformed to Hardy-Weinberg equilibrium in each cohort.The aim of this study was to explore the impact of KCNQ1 variants on the responses to oral antidiabetic drugs in a Chinese study population. a 48-week randomized pharmacogenetics study compared the effects of repaglinide and rosiglitazone in 209 newly diagnosed patients with type 2 diabetes. in the repaglinide cohort, individuals who were rs2237892 TT homozygotes exhibited lower 2-h glucose levels and significantly higher cumulative attainment rates of target 2-h glucose levels (P log-rank = 0.0383) than the C allele carriers; patients with a greater number of rs2237892 C alleles showed larger augmentations in both fasting insulin and homeostasis model assessment of insulin resistance (homair) (P = 0.0166 and 0.0026, respectively); moreover, the rs2237895 C allele was also associated with greater increments in both fasting insulin and homa-ir (P = 0.0274 and 0.0259, respectively). in contrast, only an association between rs2237897 and decrease in 2-h glucose levels was detected in the...

The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3=-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3=-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1.