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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.