Implementation of CLIP-seq technique and evaluation of mRNAs-target by protein Caprin-1

• Grant number: 14/20174-6
• Support Opportunities: Scholarships in Brazil - Master
• Start date: December 01, 2014
• End date: February 29, 2016
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Katlin Brauer Massirer
• Grantee: Natacha Azussa Migita
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 12/00195-3 - Post-transcriptional regulatory networks mediated by RNA binding proteins in human pluripotent stem cells, AP.JP

Abstract

RNA Binding Proteins (RBPs) post-transcriptionally regulate a large number of genes. RBPs bind to several cellular mRNAs and regulate splicing, localization, stabilization and degradation of transcripts, and also acting as guides in gene regulation by microRNAs. Mutations or alterations in the regulation of RBPs into the mRNAs-target are associated with disease and failures in maintaining stem cell pluripotency and processes involved in cell differentiation. However, the RBPs mechanism of action and their mRNAs-targets are largely unknown. Thus, this study aims to identify mRNA-targets bound by protein Caprin-1 (or Rng-105) and to define the binding sites of the protein in mRNAs. Caprin-1 is a cytoplasmic phosphoprotein that has RGG domain, typical of RBPs. This protein captures mRNAs on granules complexes (RNA granules or stress granules) located in the cells cytoplasm that can be quickly recruited for protein synthesis. Caprin-1 is highly expressed in neural tissues and appears to have a role in locally translate mRNAs far away from the cell body in the dendrites, and thus regulate synaptic plasticity. For this study, we will employ the CLIP-seq (Crosslinking and Immunoprecipitation of RBPs followed by RNA-seq) technique, a large-scale method to study protein-mRNAs binding with the introduction of RNA-seq. Basically, CLIP-seq consists in performing crosslinking by UV irradiation of cells, immunoprecipitation of the protein-mRNAs complex, fragmentation and selection of mRNAs bound to protein, followed by next-generation sequencing. The CLIP-seq data will be evaluated by computational tools to identify the set of mRNAs-target and the likely binding sites, defined by regions of high density on transcriptome sequences. We will utilize overexpression of Caprin-1 in HEK293 cells (Human Embryonic Kidney 293) for targets discovery by CLIP and for target validation. We aim to define the mRNAs regulation by Caprin-1 in cell granules and validate post-transcriptional regulatory events dependend on this RBP. (AU)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

MIGITA, Natacha Azussa. Profile of Caprin-1 RNA-targets. 2016. Master's Dissertation - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.