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      HITS-CLIP yields genome-wide insights into brain alternative RNA processing

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          Summary

          Protein-RNA interactions play critical roles in all aspects of gene expression. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high throughput sequencing of RNA isolated by cross linking immuno precipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova2 revealed extremely reproducible RNA binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3′ UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

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          Most cited references41

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          RNA regulons: coordination of post-transcriptional events.

          Jack Keene (2007)
          Recent findings demonstrate that multiple mRNAs are co-regulated by one or more sequence-specific RNA-binding proteins that orchestrate their splicing, export, stability, localization and translation. These and other observations have given rise to a model in which mRNAs that encode functionally related proteins are coordinately regulated during cell growth and differentiation as post-transcriptional RNA operons or regulons, through a ribonucleoprotein-driven mechanism. Here I describe several recently discovered examples of RNA operons in budding yeast, fruitfly and mammalian cells, and their potential importance in processes such as immune response, oxidative metabolism, stress response, circadian rhythms and disease. I close by considering the evolutionary wiring and rewiring of these combinatorial post-transcriptional gene-expression networks.
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            CLIP identifies Nova-regulated RNA networks in the brain.

            Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP).Thirty-four transcripts were identified multiple times by Nova CLIP.Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition.Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets.Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.
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              Splicing in disease: disruption of the splicing code and the decoding machinery.

              Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases, and recent studies have provided new insights into the mechanisms by which these effects occur. An unexpectedly large fraction of exonic mutations exhibit a primary pathogenic effect on splicing. Furthermore, normal genetic variation significantly contributes to disease severity and susceptibility by affecting splicing efficiency.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                0028-0836
                1476-4687
                6 October 2008
                2 November 2008
                27 November 2008
                27 May 2009
                : 456
                : 7221
                : 464-469
                Affiliations
                [1 ]Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave, NY, NY 10021 USA
                [2 ]MRC Laboratory of Molecular Biology, Cambridge, England
                [3 ]Expression Research, Affymetrix, Inc. Santa Clara, CA 95051
                [4 ]Biocomputing, Information Technology, The Rockefeller University, 1230 York Ave, NY, NY 10021 USA
                Author notes

                Author Contributions

                D.D.L. and R.B.D. wrote the paper. D.D.L., A.M., and J.J.F. did the biochemical and CLIP experiments. J.U. and M.K. developed ASPIRE2 and analyzed exon junction array data. D.D.L., S.W.C., X.W. and R.B.D. did bioinformatic analysis. D.D.L, J.C.D. and R.B.D. analyzed the data. T.A.C., A.C.S. and J.E.B. developed the exon junction microarray.

                Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests, except that TAC, ACS, and JEB are employees of Affymetrix, Inc. Correspondence and requests for materials should be addressed to R.B.D. ( darnelr@ 123456rockefeller.edu ).
                Article
                nihpa72490
                10.1038/nature07488
                2597294
                18978773
                9dc4d133-9c7a-4376-b137-634b43b91a5a
                History
                Funding
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS040955-05 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS034389-13A1 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS034389-12 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS034389-11 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS034389-10 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: R01 NS034389-09 ||NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Funded by: Howard Hughes Medical Institute
                Award ID: ||HHMI_
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