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Doctoral Thesis
DOI
Document
Author
Full name
Juliana da Cruz Corrêa
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Haddad, Luciana Amaral (President)
Carramaschi, Lygia da Veiga Pereira
Glezer, Isaias
Mingroni Netto, Regina Celia
Reis, Eduardo Moraes Rego
Title in Portuguese
Investigação de elementos regulatórios do éxon 14 do gene Fmr1 e análise de expressão de suas isoformas
Keywords in Portuguese
Expressão gênica
Gene FMR1
Isoformas
Splicing
Abstract in Portuguese
A proteína do retardo mental do X frágil (FMRP), codificada pelo gene do Retardo Mental do X Frágil (do inglês, Fragile Mental Retardation 1, FMR1) associa-se a RNA e transita entre o núcleo celular, grânulos citoplasmáticos e polirribossomos. No SNC, tem um importante papel como reguladora traducional atuando na maturação e eliminação sináptica. A exclusão do éxon 14 de transcritos do FMR1, associada ao uso do terceiro sítio de splicing do éxon 15 do FMR1, acarreta mudança da fase de leitura dos códons subsequentes, produzindo potencialmente isoformas da FMRP com nova região C-terminal, sem o sinal de localização nuclear e o domínio RGG, principal efetor de sua associação a RNAs. Sua importância funcional ainda não foi estudada. Neste trabalho, avaliamos por RT-qPCR a expressão dos transcritos que incluem e excluem o éxon 14 do Fmr1 no SNC de ratos e identificamos o córtex cerebral no décimo nono dia embrionário (E19) e no segundo dia pós-natal (P2) como tecido e fases do desenvolvimento em que são observados transcritos do Fmr1 com junção entre o éxon 13 e o terceiro sítio de splicing do éxon 15. Demonstramos que transcritos com essa junção exônica associam-se à proteína 4E de iniciação da tradução de eucarioto (eIF4E), indicando sua estabilidade no citoplasma. Geramos um anticorpo que identifica proteína de fusão com nova região C-terminal da FMRP. Paralelamente, a triagem por elementos em cis (transcritos) e fatores proteicos em trans trouxeram candidatos a inibirem o splicing do éxon 14 do FMR1
Title in English
Search for elements regulating FMR1 exon 14 alternative splicing and isoform analyses
Keywords in English
Gene expression
Gene FMR1
Isoform
Splicing
Abstract in English
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
 
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Release Date
2019-08-31
Publishing Date
2014-09-05
 
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