Expression and sub-cellular localization of intronic noncoding RNAs by in situ fluorescence hybridization (RNA-FISH)

• Grant number: 08/03665-5
• Support Opportunities: Scholarships abroad - New Frontiers
• Start date: September 01, 2008
• End date: August 31, 2009
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Eduardo Moraes Rego Reis
• Grantee: Eduardo Moraes Rego Reis
• Host Investigator: David L. Spector
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Cold Spring Harbor Laboratory (CSHL), United States

Abstract

Recent studies have shown that most of the eukaryotic genome is transcribed as noncoding RNAs (ncRNAs). Although it is clear that different classes of ncRNAs play fundamental roles in the control of gene expression, only a small fraction of the noncoding fraction of the transcriptome has been functionally characterized. This research proposal will employ molecular and cellular biology approaches to identify novel noncoding RNAs involved in the regulation of gene expression, based on the observation of a nuclear-restricted expression pattern, localized to discrete foci that interact closely with nuclear sub-structures that participate in RNA processing and/or chromatin remodeling. In particular, we will focus in a class of long, unspliced ncRNAs transcribed within intronic regions of the human genome, which was recently identified in our research group [Reis et al., 2004; Nakaya et al., 2007]. Sub-cellular fractionation and subsequent hybridization with high-density custom oligoarrays will be used to select nuclear retained intronic ncRNAs. Screening of nuclear intronic ncRNAs expressed in discrete foci will be pursued employing RNA-FISH. Co-localization of nuclear ncRNAs with nuclear sub-structures involved in RNA processing will be made combining RNA-FISH and immunofluorescence experiments. The set of proposed experiments will contribute to advance current knowledge on the mechanisms of biogenesis and processing of intronic ncRNAs, as well as on their possible roles in the modulation of gene expression in human tissues under normal and pathological conditions. (AU)