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

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          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) 14, 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, 79, 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) Ser 5 phosphatase Ssu72 7, 1017 and a CTD Ser 5 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 pSer 5-Pro 6 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.

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          Molecular architecture of the human pre-mRNA 3' processing complex.

          Pre-mRNA 3' end formation is an essential step in eukaryotic gene expression. Over half of human genes produce alternatively polyadenylated mRNAs, suggesting that regulated polyadenylation is an important mechanism for posttranscriptional gene control. Although a number of mammalian mRNA 3' processing factors have been identified, the full protein composition of the 3' processing machinery has not been determined, and its structure is unknown. Here we report the purification and subsequent proteomic and structural characterization of human mRNA 3' processing complexes. Remarkably, the purified 3' processing complex contains approximately 85 proteins, including known and new core 3' processing factors and over 50 proteins that may mediate crosstalk with other processes. Electron microscopic analyses show that the core 3' processing complex has a distinct "kidney" shape and is approximately 250 A in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3' processing complex.
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            Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis.

            Formation of mRNA 3' ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3' ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.
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              Protein factors in pre-mRNA 3'-end processing.

              Most eukaryotic mRNA precursors (premRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3'-end. Processing at the 3'-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3'-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Additional protein factors include poly(A) polymerase, poly(A)-binding protein, symplekin, and the C-terminal domain of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA, cleavage factor IB, and cleavage and polyadenylation factor.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                2 February 2011
                22 September 2010
                7 October 2010
                7 April 2011
                : 467
                : 7316
                : 729-733
                Affiliations
                [1 ] Department of Biological Sciences, Columbia University, New York, NY 10027, USA
                Author notes
                Correspondence and requests for materials should be addressed to L.T. ( ltong@ 123456columbia.edu )
                [2]

                Present address: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China.

                [*]

                These authors contributed equally to this work.

                Article
                nihpa226657
                10.1038/nature09391
                3038789
                20861839
                b742498b-a276-4633-a9fe-a00d6212931d

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM077175-04 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM028983-31 ||GM
                Categories
                Article

                Uncategorized
                pre-mrna processing,transcription,rna polymerase ii,protein phosphatase,peptidyl-prolyl isomerase,protein complex

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