Search for elements regulating FMR1 exon 14 alternative splicing and isoform analyses

Author(s)

Juliana da Cruz Corrêa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2014-06-06

Examining board members

Luciana Amaral Haddad; Lygia da Veiga Pereira Carramaschi; Isaias Glezer; Regina Celia Mingroni Netto; Eduardo Moraes Rego Reis

• Advisor: Luciana Amaral Haddad

Abstract

Fragile X Mental Retardation Protein (FMRP), encoded by the Fragile Mental Retardation 1 (FMR1) gene, is an RNA-binding protein with nucleus-to-cytoplasm shuttling, and polyribosome association. In the central nervous system (CNS), FMRP is a translation regulator with important roles in synaptic maturation and elimination. In primary FMR1 transcripts, exon 14 skipping followed by selection of the exon 15 third splicing acceptor site, shifts the open reading frame of the downstream codons creating a putative FMRP isoform with a novel C-terminus, which lacks the nuclear localization signal and the major RNA binding domain, RGG box. The relevance of such isoform is as yet unknown. In the present study, we assessed, by RT-qPCR, the expression of rat Fmr1 transcripts in the CNS, relative to the inclusion of exon 14. We identified the cerebral cortex in the nineteenth embryonic day (E19) and the second postnatal day (P2) as the most prominent sources of transcripts bearing a splicing junction between exon 13 and the third splicing acceptor site in exon 15. Those transcripts are associated with the eukaryotic translation initiation factor 4E (eIF4E), indicating its cytoplasmic stability. We generated an antibody that recognizes a fusion protein carrying the novel FMRP C-terminus. Concurrently, a search for ci (transcript) and trans (protein) elements identified putative inhibitors of FMR1 exon 14 splicing (AU)