Functional analyses on FMRP isoforms displaying the long, variable loop of KH2 domain

• Grant number: 11/14329-9
• Support Opportunities: Regular Research Grants
• Start date: December 01, 2011
• End date: February 28, 2014
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Luciana Amaral Haddad
• Grantee: Luciana Amaral Haddad
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Luis Eduardo Soares Netto ; Luiz Roberto Giorgetti de Britto ; Silvana Chiavegatto

Abstract

Fragile Mental Retardation Protein (FMRP), the product of Fragile Mental Retardation 1 (FMR1)gene, is an RNA-binding protein with significant expression in the brain, notably in the cerebral cortex, hippocampus, and cerebellum. Fmr1 transcripts are subjected to alternative splicing yielding 20 possible, non-redundant FMRP isoforms, not yet characterized. FMRP second KH domain (KH-2) has been implicated in ribosome binding. The length of its variable loop may be modulated by Fmr1 exon-12 alternative exclusion. We previously demonstrated that the expression of rat Fmr1 exon 12 is regulated along the early postnatal development of brain frontal córtex. We developed an antibody (3460) that detects the segment encoded by exon 12 in endogenous isoforms of FMRP (FMRP+12ISO). 3460 analyses indicated that those isoforms have an expression peak in cerebral cortex that precedes synaptic maturation. However, the mechanism of this regulation in the central nervous system (CNS) is as yet unknown. In order to understand how Fmr1 exon 12 splicing is regulated, we developed a mini-gene clone containing partial genomic Fmr1 sequence. Moreover, it is a possibility that FMRP+12ISO ribonucleoprotein complex mediates different protein or RNA interactions by FMRP KH-2 long variable loop.We here propose (i) to assess the composition of FMRP+12ISO-containing molecular complexes in cerebral cortex by immunoprecipitation and gel filtration followed by protein identification by mass spectrometry analyses; (ii) to perform immunohistochemistry analyses of embryonic rat brain to gain an overview of FMRP+12ISO in earlier phases of development not yet evaluated; and (iii) to analyze some phenotypic aspects of in vitro neuronal differentiation in neurosphere cells with Fmr1 exon 12 expression knocked down by RNAi. Finally, we aim to search for Fmr1 exon 12 splicing activators by affinity precipitation assays between biotinylated transcripts and nuclear extract protein from cerebral cortex. These assays will be performed with transcripts from the wild-type and mutated clones, the latter lacking an intronic segment that harbors splicing enhancer candidate sequences. Precipitated proteins will be evaluated by mass spectrometry and comparatively analyzed between both conditions. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CORREA-VELLOSO, JULIANA C.; LINARDI, ALESSANDRA M.; GLASER, TALITA; VELLOSO, FERNANDO J.; RIVAS, MARIA P.; LEITE, RENATA E. P.; GRINBERG, LEA T.; ULRICH, HENNING; AKINS, MICHAEL R.; CHIAVEGATTO, SILVANA; et al. Fmr1 exon 14 skipping in late embryonic development of the rat forebrain. BMC NEUROSCIENCE, v. 23, n. 1, p. 15-pg., 2022-05-31. (18/07366-4, 17/06100-8, 19/10868-4, 11/14329-9, 09/01333-8)