Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5′ and 3′ splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5′ and 3′ splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5′ and 3′ splice site selection.

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5′ splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5′ splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5′ end, suggesting that other factors than the U1 snRNA are involved in 5′ splice site recognition. The importance of a consecutive complementarity between the 5′ splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3′ splice site of the regulated intron. We now report that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5′ splice site is required for efficient splicing inhibition by SXL. Psoralen-mediated crosslinking experiments suggest that SXL binding to this uridine-rich sequence inhibits recognition of the 5′ splice site by U1 snRNP in HeLa nuclear extracts. We also show that SXL interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5′ splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 5′ splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL.

Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein–protein interactions. It has been previously shown that, in yeast, communication mediated by RNA–RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5′ splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement.

The inactivity or occlusion of the HIV-1 poly(A) signal when in the 5′ long terminal repeat (LTR) has been mechanistically investigated. First we show that neither the homologous HIV-1 promoter nor the close proximity of this RNA processing signal to the transcript initiation site is required for the occlusion effect. Instead we demonstrate that the major splice donor (MSD) site positioned about 200 bp downstream maintains the poly(A) site in an inactive state. Although mutation of MSD results in activation of the 5′ LTR poly(A) signal, this effect can be suppressed by targeting U1 snRNAs near to the mutated MSD by base pairing. We show that hybrid U7-U1 snRNAs can also suppress the poly(A) signal and that this suppression is dependent on the U1 stem-loop 1. In particular the binding site for the U1 snRNP protein 70K that binds to the loop structure of stem-loop 1 is associated with poly(A) site occlusion. These experiments were carried out with an HIV-1 proviral construct and as such emphasize the physiological importance of this splice donor–poly(A) site interaction.

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-U1A and U2 snRNP-U2B″. The structure and sequence of SNF, U1A, and U2B″ are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B″ binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf 's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the “RNA-specificity” domain is necessary for SNF's dual function whereas the mixed motif in the U2A′-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5′ splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5′ splice site and by an exonic enhancer differ mechanistically.