Benoit Chabot

Modulation of exon skipping by high‐affinity hnRNP A1‐binding sites and by intron elements that repress splice site utilization

The RNA‐binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre‐mRNA. Each intron flanking exon 7B contains a high‐affinity A1‐binding site. The A1‐binding elements promote exon skipping in vivo, activate distal 5′ splice site selection in vitro and improve the responsiveness of pre‐mRNAs to increases in the concentration of A1. Whereas the glycine‐rich C‐terminal domain of A1 is not required for binding, it is essential to activate the distal 5′ splice site. Because A1 complexes can interact simultaneously with two A1‐binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1‐binding sites in various pre‐mRNAs, an A1‐mediated change in pre‐mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3′ splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.

Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5′ splice sites. We find that a recombinant GST-MS2 protein interferes with 5′ splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5′ splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism.

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5 splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5 splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5 splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5 splice site usage in an equivalent manner. Second, increasing the distance between the internal 5 splice site and flanking A1 binding sites does not compromise activation of the distal 5 splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5 splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5 splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5 splice site and lead to distal 5 splice site activation.

Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5' splice site selection in the hnRNP A1 mRNA precursor.

In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7Bin vivo and distal 5′ splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5′ splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5′ splice site activation. Moreover, the ability of CE1d to stimulate distal 5′ splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5′ splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5′ splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.