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      High-throughput Methods for Dissection of Trypanosome Gene Regulatory Networks

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          Abstract

          From synthesis to decay, mRNA associates with RNA-binding proteins (RBPs) establishing dynamic ribonucleoprotein particles (RNPs). Understanding the composition and function of RNPs is fundamental to understanding how eukaryotic mRNAs are controlled. This is especially relevant for trypanosomes and related kinetoplastid parasites, which mostly rely on post-transcriptional mechanisms to control gene expression. Crucial for trypanosome differentiation, development, or even response to heat shock, RBPs are known to be essential modulators of diverse molecular processes. The recent application of large-scale quantitative methods, such as Next-Generation Sequencing (NGS) and quantitative mass spectrometry, has revealed new exciting features about the parasite RNA-related metabolism. Novel proteins carrying RNA-binding activity, including many proteins without RNA-related ontology were discovered setting a necessary groundwork to get in insights into RNA biology.

          Conclusion: This review aims to give the reader an understanding of current trypanosome RNP research, highlighting the progress made using high-throughput approaches.

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          Most cited references92

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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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            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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              RNA granules

              Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationship between different classes of these granules and discuss how spatial organization regulates messenger RNA translation/decay.
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                Author and article information

                Journal
                Curr Genomics
                Curr. Genomics
                CG
                Current Genomics
                Bentham Science Publishers
                1389-2029
                1875-5488
                February 2018
                February 2018
                : 19
                : 2
                : 78-86
                Affiliations
                [1]Zentrum fur Molekulare Biologie der Universitet Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120, Heidelberg , Germany
                Author notes
                [* ]Address correspondence to this author at the German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Tel/Fax: +49-6221-421392/+49-6221-421398; E-mails: e.erben@ 123456dkfz-heidelberg.de ; e.erben@ 123456zmbh.uni-heidelberg.de
                Article
                CG-19-78
                10.2174/1389202918666170815125336
                5814965
                29491736
                0be23d3e-c9bf-40da-93c9-75d3e73ea88c
                © 2018 Bentham Science Publishers

                This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) ( https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

                History
                : 15 November 2016
                : 22 December 2016
                : 03 February 2017
                Categories
                Article

                Genetics
                rna-binding proteins,post-transcriptional control,high-throughput,gene expression regulation,mrna-fate,rna biology

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