Worm Breeder's Gazette 12(2): 66 (January 1, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have been working on the problem of what signals an initial transcript to be trans-spliced. Our previous results (Conrad et al. 1991,Mol. Cell. Biol. 11:1921) indicated that a transcript that begins with an intron, rather than the first exon, is trans-spliced to SL1 ,and that no sequence-specific information is required for SL1 splicing. We named this 5' intron an "outron". The requirement for the splice-acceptor site at the outron/exon border is a close match to the consensus: UUUCAG/Pu. Our interest now is in what is required upstream of the splice site. Our initial hypothesis is that both AU-richness and a minimum length are the primary, and perhaps sole, requirements. This expectation arises from several observations: 1) our previous results showed that an intron contains all the information necessary to signal trans-splicing, 2) no consensus sequences can be discerned from gazing at the many known outron sequences, and 3) there is a perfect splice-acceptor site in the 5' UTR of act-4 which is not trans-spliced, and its 5' UTR is only 33 nt and ~50% AU (functional outrons and introns are around 70% AU). To test the hypothesis we are creating a set of synthetic outrons to replace the rol-6 outron. These constructs can then be used to transform worms, and the resultant RNAs analyzed to determine if the particular potential outron is functional for trans-splicing.
We deleted all but the six nucleotides at the 5' end and the six at the 3' end of the (normally 173 nt) rol-6 outron. This preserves (we hope) the transcriptional start site and splice acceptor site. In place of the 161 deleted bases we inserted a 15 nt sequence containing a unique XhoI site. We called this construct OM (for minimal outron). On assaying this construct using RNA PCR, we found that its RNA product was not trans-spliced. Further constructs were created using an oligonucleotide duplex as an insert into the XhoI site. The insert was 25 nt long, with the repeated sequence A2U4 in one orientation ("[U]") and U2A4 in the other ("[A]"). A triple-insert, S'[U][U][A]3', gave strong trans-splicing, with little unspliced product accumulating. Single inserts (both [A] and [U]) also were trans-spliced, but unspliced product also accumulated,so we presume the trans-splicing was less efficient with these shorter outrons. A 5'[U][U]3' double-insert gave an intermediate trans-spliced-to-unspliced ratio. Almost all trans-splicing was to SL1 ,which provides the strongest support yet that SL1 splicing is the default mode. We conclude that SL1 trans-splicing can be signalled simply by the presence at the 5' end of a pre-mRNA of an AU-rich sequence followed by a splice-acceptor site.
A final point is that all of these constructs had to be co-injected with a wild-type rol-6 construct ( pRF5 ).No stable roller strains were obtained with any of the OM-derived constructs, although roller F1 swere. (We can distinguish the gene products of pRF5 and the OM constructs because the latter were marked by a set of codon-conserving nucleotide changes, a 5-base change in a 7-base stretch). This raises some interesting questions about a possible role for the portion of rol-6 that contains the outron sequence in promoter function or in stabilization of the pre-mRNA.