Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with ~70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5′ splice site located downstream of a weakened 5′ splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5′ splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5′ splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5′ splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate limiting step(s) in splicing can lead to dramatic shifts in splice site choice.

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A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans ( Communicated by Lester 0. Krampitz, July 25, 1988 (received for review June 6, 1988 ABSTRACTThe mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.Evidence suggests that intermolecular (trans) splicing is used in a variety of organisms during the maturation of some mRNAs. This is particularly clear for trypanosomatid protozoans, where all mRNAs contain a common leader derived from a small nonpolyadenylylated miniexon transcript (for review, see ref. 1). A trans-splicing mechanism of leader addition is supported by the primary structure of the miniexon transcript and the existence of appropriate branched intermediates (2, 3). Recent observations indicate that transsplicing might also be used in the formation of mRNA for chloroplast ribosomal protein S12 (4) and in the maturation of certain actin mRNAs in Caenorhabditis elegans (5).In C. elegans, mRNAs derived from three of four actin genes contain a 22-nucleotide (nt) leader sequence that is not encoded within 15 kilobases (kb) of the actin genes. This leader sequence is found as the first 22 nt of an abundant 100-base RNA transcribed from within the 5S rRNA gene cluster (5). Several lines of evidence, including the demonstration of branched intermediates containing a portion of the 100-nt RNA, suggest that the 22-nt leader is acquired by trans-splicing (5, 16). In contrast to the situation in trypanosomes, only a subset of C. elegans mRNAs appear to contain the trans-spliced leader. Furthermore, because C. elegans actin genes contain multiple introns, trans-splicing apparently occurs in conjunction with conventional cis-splicing. As discussed by Krause and Hirsh (5) the use of trans-splicing in C. elegans raises the possibility that this mechanism could be widespread in eukaryotes and may be a regulatory mechanism in gene expression.We have recently described the isolation and characterization of cDNA and genomic clones encoding a 63-kDa protective antigen in the human parasitic nematode Brugia malayi, the causative agent of lymphatic filariasis (6, 7).Nuclease protection and primer-extension experime...

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5' exon, the SL, is affixed to the 5'-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.