We have used the parasitic nematode, Ascaris, as a model system for the biochemical analysis of nematode gene expression. Cell free extracts derived from synchronously developing Ascaris embryos have proven remarkably versatile for reproducing a variety of cellular processes in vitro. These extracts catalyze accurate transcription of RNA polymerase II and RNA polymerase III genes as well as cis- and trans-splicing.
Nematodes are among the few organisms which are known to carry out intermolecular (trans) RNA splicing. Ascaris extracts are unique in their ability to catalyze trans-splicing in vitro. In these extracts, the spliced leader sequence is donated to a synthetic pre-mRNA acceptor from either endogenous SL RNA or from synthetic SL RNA. Detailed analysis of reaction intermediates and products revealed that trans-splicing is mechanistically analogous to cis-splicing. However, these reactions differ in their cofactor requirements. Cis-splicing requires intact U1, U2 and U4/U6 snRNPs. Trans-splicing, however, does not require an intact U1 snRNP.
Our analyses have revealed that the SL RNA participates in trans-splicing as an Sm snRNP. However, much of the primary sequence of the SL RNA is dispensable for its function. In particular, intramolecular base-pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro. This interaction was proposed to substitute in trans-splicing for the base pairing between U1 snRNA and conventional cis-splice sites. Furthermore, the 22 nucleotides SL sequence is dispensable for trans-splicing and can be replaced by artificial exons of 2 to 246 bases. Strikingly, the essential properties of the SL RNA are encoded within a short sequence surrounding the SL RNA Sm binding site. This sequence alone can convert a fragment of the U1 snRNA into a functional SL RNA.
Ascaris extracts are also unusual in their ability to efficiently transcribe the U-snRNA genes. We have analyzed the transcriptional control elements for one U-snRNA gene, the SL RNA gene, in detail. Remarkably, the sequences which signal termination of SL RNA transcription reside within the SL RNA coding sequence, and internal elements, particularly the SL sequence, are also essential for transcription initiation.