Caractérisation de deux élements introniques modulant l'épissage alternatif de l'ARN pré-messager du gène de hnRNP A1
Alternative splicing of the pre-mRNA of hnRNP A1 leads to the production of two protein isoforms, hnRNP A1 or hnRNP A1[superscript B]. Previous studies comparing the human and mouse hnRNP A1 genes revealed several highly conserved intronic sequences flanking the alternative exon 7B. These conserved regions were divided into ten conserved elements (CE1 to CE10) allowing for individual study. The studies presented here concern two conserved sequence elements: CE3 and CE-D. CE3 is a large conserved region located directly upstream of the 3' splice site of exon 7B and extends for 60 nucleotides. We mapped the branch site of intron 7A within a region 16 to 25 nucleotides upstream of the 3' splice site. However, the positioning of this branch site appears sub optimal. We focused on the sequences of CE3 extending 40 nucleotides upstream of the branch site. Using an in vitro splicing assay with a pre-mRNA carrying two 3' splice sites in competition for a single 5' splice site, we found that an excess of competitor RNA containing the sequences upstream of the branch site promoted an increase in the use of the 3' splice site of exon 7B. These results suggest that the sequences modulate the use of the 3' splice site and that a nuclear factor is involved in its activity. The use of the same competitor RNA in a UV cross-linking assay showed that the RNA could specifically compete for the binding of a 60-kDa protein. An attempt to identify this factor using RNA affinity chromatography performed on a HeLa nuclear extract revealed that several nuclear factors interacted with this 40 nucleotide sequence. In vitro splicing assays performed in the depleted HeLa nuclear extract resulted in a small increase in splicing to the proximal 3' splice site. These results suggest that nuclear factors interact with sequences upstream of the branch site within the CE3 sequences and that their binding can suppress the use of the 3' splice site of exon 7B. Previous studies of a conserved element (CE1) within the intron separating exon 7 from 7B had identified a high-affinity A1 binding site (CE1a) which promoted exon skipping in vivo and distal 5' splice site activation in vitro. The effects of the 17 nucleotide long CE1a element was not as dramatic as the effects observed with the complete element (CE1). My study aimed at determining the contribution of other sequences within CE1 led to the identification of a region (CE-D) flanking CE1a that has a similar activity on 5' splice site selection. The interaction of CE-D with members of the hnRNP A1/A2 family of proteins and the observation that a transcript containing the CE-D element is more sensitive to the presence of hnRNP A1 suggests that the activity of the CE-D element is mediated by hnRNP A1. The presence of both CE1a and CE-D elements allows for the full restoration of CE1 activity. These results suggest that the CE1 element is composed of two A1 binding sites which modulate the selection of 5' splice sites.