Identify and characterize proteins containing the RNA-binding motifs RNP1 and RNP2 in the presynaptic terminals of neurons from squid photoreceptors

• Grant number: 11/13635-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: October 01, 2011
• End date: September 30, 2014
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Roy Edward Larson
• Grantee: Diego Torrecillas Paula Lico
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

SummaryDuring my doctoral thesis I identified and partially characterized a novel 65 kDa RNA binding protein (p65) in presynaptic terminals of the Brazilian squid Loligo plei. Based on sequences obtained by mass spectroscopy from purified p65, two polyclonal antibodies raised in rabbits were immunoreactive towards the consensus sequences for RNA binding (the RNA recognition motifs, referred to as RNA binding protein domains RBP1 and RBP2). One of these antibodies labeled p65 on western blots, thus confirming that the original and new antibody target the same protein. In addition, the antibodies revealed 3-4 other immunoreactive bands on western blots, possible isoforms and/or homologous proteins having the RBP 1 and 2 motifs, indicating that we are dealing with a family of proteins. Also, in silica analyses based on the mass spectrometry data, indicated that p65 is a member of a group of RNA binding proteins of the subtype hnRNP A/B. These data form the basis of the present project.In this project we intend to clone, sequence, and characterize p65 starting from the sequence data already obtained. Our evidence indicates that p65 is part of a ribonucleoprotein complex encountered in the presynaptic region of squid neurons; thus, we intend to isolate and characterize the components of this complex in synaptosomes from the optic lobes of squid. Once isolated, the particles will be analyzed by mass spectrometry to identify protein components and in silica analyses to formulate hypotheses as to function, as well as to generate antibodies and molecular probes as tools toward dissecting function. Other proteins of this subgroup of RNA binding proteins encountered will be objects of further study using similar approaches as applied to p65.