Molecular and functional characterization and RNAs enriched in the nucleus of mammalian cells

• Grant number: 11/51803-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2012
• End date: March 31, 2015
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Eduardo Moraes Rego Reis
• Grantee: Ana Carolina Ayupe de Oliveira Beckedorff
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Evidence from the literature indicate that a significant fraction of the polyadenylated RNAs produced in eukaryotic cells accumulate in the nucleus. The functionality of coding and noncoding RNAs that accumulate in a stable form the nucleus, and the contribution of this transcript pool to gene expression control are not well known. In this project, we will investigate the structure, biogenesis, processing and association in ribonucleoproteins (RNPs) of cellular RNAs that are enriched in nucleus of human cell lines. Initially we will characterize three mRNAs identified in previous work performed in our group that associate with nuclear protein PSP1 and that are enriched in the nucleus of human cells lines (SLC38A1, SLC38A2 e PTP4A1). We will investigate the contribution of post-transcriptional processing mechanisms such as adenosine-inosine editing and/or alternative splicing in the nuclear retention mechanism of selected candidates. We will conduct RNA pull-down assays to identify proteins associated with candidate RNAs that may be involved in the nuclear retention mechanism. To that end, co-purified proteins that associate with RNAs in ribonucleoprotein (RNP) complexes will be identified by tandem mass spectrometry (MS-MS). We will also evaluate the dynamics of RNA nuclear retention during cellular differentiation and the relevance of this process to the maintenance of the pluripotency state using a murine model of neural differentiation (P19), by profiling the nuclear and cytoplasmic transcriptome of undifferentiated P19 cells and cells differentiated in neurons or glia. The set of proposed experiments will provide new information about the molecular mechanisms involved in RNA nuclear retention and the functional relevance of this mechanism to fine tuning of gene expression control in mammalian cells. (AU)