RESUMO
Out of the loop: Do the proteins bound to an enhancer site on pre-mRNA interact directly with the splice site by diffusion (looping), as is generally accepted, or does the intervening RNA play a role? By inserting a PEG linker between an enhancer sequence and alternative splice sites, the interaction of these two elements can be studied. Intervening RNA was essential for the enhancer activity, which rules out the looping model.
Assuntos
Elementos Facilitadores Genéticos , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Sequência de Bases , Química Click , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5' end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.