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Pre-mRNA splicing involves recognition of a consensus sequence at the 5 splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5 SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5 SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs. M ost eukaryotic genes contain introns whose precise removal by the pre-mRNA splicing machine is an essential step in gene expression. The accuracy and efficiency of premRNA splicing is attributed to a number of transacting factors, which include the spliceosomal U small nuclear ribonucleoproteins (snRNPs) and several non-snRNP protein splicing factors, as well as to cis-acting sequence elements. The latter include 5Ј and 3Ј splice sites (SSs), a branch point, a polypyrimidine tract, and splicing enhancer and silencer sequence elements. A key step in pre-mRNA splicing involves the recognition and selection of a consensus sequence AG͞GTRAGT (in mammals, where R denotes purine and͞denotes the splice junction) at the 5Ј SS (1-3). Frequently, however, sequences that comply with the consensus are not selected for splicing (4). We refer to such 5Ј consensus sequences as latent 5Ј SSs, to splicing events in which they are used as latent splicing, and to the resulting mRNA as latent RNA.Latent 5Ј SSs are highly abundant in pre-mRNAs. In a survey of a database consisting of 446 human protein-coding genes (http:͞͞www.fruitfly.org͞seqtools͞datasets͞Human͞) we identified 10,626 latent 5Ј SSs located within introns. Thus, the splicing machine must frequently discriminate between normal and latent 5Ј SSs. How this is achieved is not yet understood. A clue for a possible mechanism arises from the fact that the intron sequences upstream to 94.5% of the intronic latent sites contain at least one stop codon in the reading frame determined by the bona fide upstream exons. This general finding conforms with the idea that the cell's nucleus harbors a checking mechanism that is capable of recognizing premature stop codons in premRNAs, and consequently aborting splicing at downstream latent 5Ј SSs (5).A straightforward prediction of this model is that the removal of in-frame stop codons would render downstream latent 5Ј SSs active in splicing. As a test case we chose the gene...

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We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) In view of the proposed role of the SR proteins in premRNA splicing, this family of proteins is expected to be associated with the RNA processing machinery in vivo. PremRNA splicing in vivo apparently occurs on structures more complex than individual spliceosomes. This should not be surprising, because the splicing machinery of a living cell is expected to package and process multi-intronic pre-mRNAs, rather than the mono-intronic pre-mRNAs, which are typically used to assemble spliceosomes in vitro. Indeed, we have shown that several specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A) + RNA, are packaged in compact large nuclear RNP (lnRNP) particles that invariably sediment at the 200S region in sucrose gradients (31)(32)(33)(34) 8830The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5' and 3' splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.

The effect of heat shock on the packaging and splicing of nuclear CAD pre-mRNA, a transcript expressed constitutively from a non heat-inducible promoter, was studied in vivo in Syrian hamster cells. While mild heat shock did not affect significantly the packaging of CAD RNA in 200S InRNP particles, it caused perturbation to splicing. First, the heat shock inhibited splicing of CAD pre-mRNA. Second, it affected 5' splice site selection by activating cleavage at a cryptic 5' splice site; yet ligation of the cryptic exon to the downstream proximal exon was not observed. Base complementarities of the cryptic site with U1, U5, or U6 snRNAs are comparable, or even better, than those with the neighboring normal site. Hence, the exclusion of the cryptic site under normal growth conditions cannot be attributed to weaker base pairing with these snRNAs. On the other hand, these results imply the involvement of a heat labile factor in the selection of the 5' cleavage site. The exclusion of the cryptic site at 37 degrees C and the aborted splicing at this site after heat shock may also be explained by a proposed nuclear checking mechanism that detects in-frame stop codons upstream of the 5' splice site, and aborts splicing at such sites to prevent the production of a defective message.

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RNA sequences that conform to the consensus sequence of 5 splice sites but are not used for splicing occur frequently in protein coding genes. Mutational analyses have shown that suppression of splicing at such latent sites may be dictated by the necessity to maintain an open reading frame in the mRNA. Here we show that stop codon frequency in introns having latent 5 splice sites is significantly greater than that of introns lacking such sites and significantly greater than the expected occurrence by chance alone. Both observations suggest the occurrence of a general mechanism that recognizes the mRNA reading frame in the context of pre-mRNA. Keywords: latent 5 splice sites; splice site selection; stop codons; suppression of splicing (SOS); translatabilityIn a recent paper, Zhang et al. (2003) presented a computational analysis that attempted to challenge the idea that the translatability of an exon can influence its splicing. This idea has been recently supported by a number of studies: Wang et al. (2002) and Mendell et al. (2002) demonstrated the occurrence of nonsense-associated alternative splicing in mutated genes, and a study from our laboratory demonstrated that splicing at intronic latent 5Ј splice sites is suppressed in wild-type genes when in-frame stop codons occur between the latent and the upstream normal (authentic) 5Ј splice site.Latent 5Ј splice sites are defined as sequences that conform to the canonical 5Ј splice site consensus sequence but, normally, are not used for splicing. Latent sites appear to be highly abundant in the genome, particularly in introns of protein-coding genes (see below). To explain the phenomenon of suppression of splicing (SOS) at latent 5Ј splice sites, we proposed that the necessity to maintain the translatability of mRNAs, by avoiding the inclusion of premature termination codons in them, could serve as a criterion that differentiates normal 5Ј splice sites from latent ones (Miriami et al. 1994). We substantiated this proposal by showing, in two gene systems, that an intronic latent 5Ј splice site can be activated if all upstream stop codons, which are in the reading frame of the upstream exon, are eliminated by point or frame-shift mutations . We also validated, by three criteria, the generality of the translatability hypothesis in a computerized genomic survey of a human database of 2206 introns, 1496 of which contain a total of 10,490 latent 5Ј splice sites and 710 of which are devoid of such sites. First, of the 1496 introns with latent 5Ј splice sites, 1359 (90.8%) have at least one in-frame stop codon upstream of the most 3Ј latent site. Second, in-frame stop codons occur upstream of 10,045 (95.8%) of the 10,490 latent 5Ј splice sites. Third, the density of in-frame stop codons in the 1496 introns with latent 5Ј splice sites is significantly higher than that in the 710 introns devoid of latent sites (0.0484 versus 0.0338 per effective number of codons; P < 0.001; Miriami et al. 2002). Zhang et al. (2003) challenged the translatability concept because th...