AN INTRON ELEMENT MODULATING 5'-SPLICE-SITE SELECTION IN THE HNRNP A1PRE-MESSENGER-RNA INTERACTS WITH HNRNP A1

The hnRNP Al pre-mRNA is alternatively spliced to yield the Al and Alb mRNAs, which encode proteins differing in their ability to modulate 5 ' splice site selection, Sequencing a genomic portion of the murine Al gene revealed that the intron separating exon 7 and the alternative e xon 7B is highly conserved between mouse and human, In vitro splicing assays indicate that a conserved element (CE1) from the central portio n of the intron shifts selection toward the distal donor site when pos itioned in between the 5' splice sites of exon 7 and 7B, In vivo, the CE1 element promotes exon 7B skipping, A 17-nucleotide sequence within CEI (CE1a) is sufficient to activate the distal 5' splice site. RNase T-1 protection/immunoprecipitation assays indicate that hnRNP Al bind s to CE1a, which contains the sequence UAGAGU, a close match to the re ported optimal Al binding site, UAGGGU. Replacing CE1a by different ol igonucleotides carrying the sequence UAGAGU or UAGGGU maintains the pr eference for the distal 5' splice site, In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site, In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficienc y of Al binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to compet ing 5' splice sites, as judged by oligonucleotide-targeted RNase H pro tection assays, Our results suggest that hnRNP Al modulates splice sit e selection on its own pre-mRNA without changing the binding of U1 snR NP to competing 5' splice sites.