Molecular and cellular characterization of neuronal hnRNPA/B proteins

• Grant number: 15/13151-2
• Support Opportunities: Regular Research Grants
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Roy Edward Larson
• Grantee: Roy Edward Larson
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Project summary. We have purified and partially characterized an RNA-binding protein of 65 kDa (p65) from optic lobes of squid (Doryteuthis pleii, South Atlantic species and Doryteuthis pealii, North Atlantic species) (Lico et al. 2010; 2015). It has been identified as a member of the heterogenous nuclear ribonucleoprotein family, subgroup A/B (hnRNPA/B). Cloning, sequencing and data mining revealed that there are at least 11 transcripts in squid that encode hnRNPA/B-like proteins with a high degree of homology. One of these transcripts, which corresponded to a protein that had physical properties that implicated it as a subunit of p65, was cloned and expressed in bacteria (referred to as hnRNPA/B-like protein 2). This recombinant protein showed propensity to oligomerize and could form an SDS-stable dimer when incubated with optic lobe extract (Lico et al., 2015). Recently, mutations in hnRNPA/B proteins have been implicated in neurodegenerative diseases by their promotion or facilitation of the formation of inclusion bodies. These facts and localization of p65 in pre-synaptic terminals led to the hypothesis that p65 participates in mechanisms of site-specific processing and/or translation of RNA, and could offer a link to understanding RNA´s role at the level of the presynaptic terminal and neurotransmission control. With the intent to identify components of RNP complexes containing p65, we will isolate RNP complexes from optic lobe extracts by centrifugation in sucrose gradients, and pulldown techniques, and identify components by mass spectrometry and antibody probes. In order to investigate localization and cellular dynamics we will apply confocal microscopy techniques to subcellular localization of endogenous proteins and recombinant proteins transfected into SH-SY5Y neuroblastoma cells, an in vitro model for neuronal differentiation and functionality. We expect that our results will contribute to an understanding of the molecular properties of p65, as well as other hnRNP proteins, and their role in the regulation of presynaptic function. (AU)