Exon definition may facilitate splice site selection in RNAs with multiple exons.

Abstract

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity.

“…In the 5Ј-splicing consensus sequence, the dinucleotide GT is the best conserved among mammals, and mutations affecting this sequence were reported to cause the inactivation of several genes associated with human diseases including phenylalanine hydroxylase (19), procollagen COL3A1 (20), and hypoxanthine guanine phosphoribosil-transferase (21). In these genes, a G to A mutation in the GT splicing recognition sequence consistently results in skipping of the entire exon preceding the mutation, supporting the current theory of exon definition for higher eukaryotes in which the splicing sites are recognized as exonic pairs as opposed to the mechanism proposed for lower eukaryotes in which the splicing sites are recognized as intronic pairs (22,23).…”

Deletion of the donor splice site of intron 4 in the glucokinase gene causes maturity-onset diabetes of the young.

“…In the MODY family F423, the abnormal 424 bp transcript results from the activation of a cryptic splice site situated in exon 4, close to and upstream (24 bp) the deleted consensus gt site, as usually reported (28,29). The sequence surrounding this cryptic site (CT gtgagg) is in agreement with the consensus sequence of5 '-splice site in mammals: AG gta (or g) agt for the positions -2 to +6 based on the beginning of the intron where the splice site is situated.…”

“…In most cases reported previously (28,29,31), when the 5 '-splice site was altered, the skipped exon was exclusively the exon located on 5' of the altered donor splice site. The mechanisms involved in family F423, leading to the skipping of the exon situated on 3' of the affected donor splice site, remain obscure.…”

Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP

“…Whether the major spliceosomal snRNAs are involved in splicing enhancement, that is, in recognition of purinerich enhancers or in bridging between enhancer-binding factors and conserved intron elements, is difficult to address rigorously in the context of the major splicing pathway because of the requirement for these snRNAs in the basal splicing reaction+ U1 snRNP has been shown to play a role in exon definition in both the major and minor splicing pathways, and it does so by binding to the downstream conventional 59 splice site (Robberson et al+, 1990;Kuo et al+, 1991;Wu & Krainer, 1996)+ A role for an snRNA in enhancer recognition has previously been suggested in the case of U1 (Watakabe et al+, 1993;Staknis & Reed, 1994;Stark et al+, 1998)+ Because the basal AT-AC splicing reaction does not require the major snRNAs, except for the U5 snRNA, nor is inactivation of the major pathway required in our in vitro system, we have exploited these unique snRNA requirements to investigate whether the snRNAs that function exclusively in the conventional pathway are required for enhancer function in the context of AT-AC splicing+…”