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      Identification of proteins and miRNAs that specifically bind an mRNA in vivo

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          Abstract

          Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

          Abstract

          The entire mRNA interactome capture has been established but identification of specific mRNA-binding proteins has been challenging. Here, the authors developed an in vivo RNA–protein crosslinking and RNA pulldown approach and characterized novel interactors of the C. elegans gld-1 mRNA, including DAZ-1 and miR-84.

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          Most cited references44

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          Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing

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            Single-copy insertion of transgenes in Caenorhabditis elegans.

            At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
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              HITS-CLIP yields genome-wide insights into brain alternative RNA processing

              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 crosslinking immunoprecipitation (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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                Author and article information

                Contributors
                kathrin.theil@gmx.de
                rajewsky@mdc-berlin.de
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                16 September 2019
                16 September 2019
                2019
                : 10
                : 4205
                Affiliations
                [1 ]ISNI 0000 0001 1014 0849, GRID grid.419491.0, Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), , Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), ; 13125 Berlin, Germany
                [2 ]ISNI 0000 0001 1014 0849, GRID grid.419491.0, Proteome Dynamics, , Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), ; 13125 Berlin, Germany
                [3 ]ISNI 0000 0004 0372 2033, GRID grid.258799.8, Laboratory of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, , Kyoto University, ; Kyoto, 606-8501 Japan
                Author information
                http://orcid.org/0000-0002-7205-6308
                http://orcid.org/0000-0002-7451-4982
                http://orcid.org/0000-0002-4785-4332
                Article
                12050
                10.1038/s41467-019-12050-7
                6746756
                31527589
                1a213ed0-7177-4744-a151-d6cf75d06f51
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 August 2018
                : 19 August 2019
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                Custom metadata
                © The Author(s) 2019

                Uncategorized
                biochemistry,rna,biological techniques,experimental organisms,mass spectrometry
                Uncategorized
                biochemistry, rna, biological techniques, experimental organisms, mass spectrometry

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