We set up an alternative splicing system in vitro in which the relative amounts of two spliced RNAs, one containing and the other lacking a particular exon, were directly proportional to the length of an inverted repeat inserted into the flanking introns. We then used the system to measure the effect of intramolecular complementarity on alternative splicing in vivo. We found that an alternative splice was induced in vivo only when the introns contained more than -50 nucleotides of perfect complementarity, that is, only when the secondary structure was much more stable than most if not all possible secondary structures in natural mRNA precursors. We showed further that intron insertions containing long complements to splice sites and a branch point inhibited splicing in vitro but not in vivo. These results raise the possibility that in cells most pre-mRNA secondary structures either are not maintained long enough to influence splicing choices, or never form at all.The splicing of different sets of exon sequences from a primary transcript is one of several strategies that eucaryotic cells use to diversify their gene products. For example, the transcripts of many genes for muscle proteins are spliced in alternative ways to generate mRNAs that encode several different forms of the proteins. Furthermore, the splicing choices for some of these (and other) gene transcripts appear to be regulated in a developmental or tissue-specific fashion (14). One presumes that the transcripts have cis-acting signals that direct splice site selection and that trans-acting factors somehow interact with those signals to regulate the selections. But the nature of such factors or signals, beyond the RNA sequences near the splice sites themselves, remains a mystery.Using an in vitro splicing system, we showed previously (23) that well-defined secondary structures inserted into a pre-mRNA changed the splicing pattern in a predictable way. For instance, when an exon was put into the loop of a hairpin by inserting long (125-nucleotide [nt]) inverted repeats into the adjoining introns, the splicing apparatus generated a novel, "jump"-spliced RNA that had the flanking exons joined directly together, leaving out the exon in the loop. To a lesser extent, the hairpin caused the same alternative splice in vivo, in transfected HeLa cells. This observation established the potential of secondary structure to influence splicing choices, but did not establish whether such a mechanism is normally used. To begin to address this question, we have measured how much complementarity is required to induce the alternative splice in vivo. MATERIALS AND METHODSAll the methods used for the in vitro and in vivo splicing assays were described previously (23). RESULTS AND DISCUSSIONMeasuring the stem length required to generate an alternative (jump) splice. Figure 1 outlines the experimental approach. We modified a plasmid template that contained the first intron and first two exons (El and E2) of the transcription unit encoding the adenovirus tripartite leader. T...
A adenovirus type 5 host range mutant (hr440) has been isolated which is defective in a splicing event required to generate the middle-sized mRNA from early region 1A. This defect has been ascribed to two adjacent nucleotide changes which lie five and six nucleotides from the 5' splice site for this mRNA (Solnick, Nature 291:508-510, 1981). One of these changes introduces an amber codon into the reading frame of the largest region 1A mRNA, resulting in the production of a truncated polypeptide. Like other region 1A mutants, hr440 is defective in the production of mRNA from early regions 2 and 3, but hr440 is unusual in that transcription from regions 1B and 4 is normal. Furthermore, although region 1B expression is unaffected, hr440 does not transform baby rat kidney cells. Therefore, expression of early region 1B is insufficient for transformation, eliminating the possibility that region 1A is required only to induce such expression.
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