Tom Blumenthal

My laboratory works on the small nematode worm, C. elegans, as a model to understand gene regulation and expression in higher animals. In the process of studying mechanisms of mRNA splicing in C. elegans, our laboratory has discovered that a significant proportion of genes in this animal are arranged and transcribed in a manner quite similar to bacterial operons. This is very unusual in eukaryotes and previously unheard of in animals. The polycistronic pre-mRNAs are converted into monocistronic units by cleavage and polyadenylation and by trans-splicing. We are interested in three general questions. 1) What mechanisms are involved in processing of the polycistronic mRNAs? 2) Do the C. elegans operons serve the purpose of co-regulating genes whose products function together, as operons do in bacteria? and 3) How and when did operons arise during eukaryotic evolution? Like genes in other animals, C. elegans genes contain introns that are spliced out by spliceosomes. However, C. elegans has another type of splicing, mechanistically quite similar to removal of introns, called trans-splicing, in which a short leader is spliced onto the 5' ends of mRNAs. Recognition of a trans-splice site requires only that the pre-mRNA have a 3' splice site with no upstream 5' splice site. Although, C. elegans has two spliced leaders, SL1 and SL2, only SL1 is utilized in such situations. SL2 is reserved for genes located in downstream positions in operons. There is a close connection between formation of the 3' end of the upstream gene and trans-splicing about 100 nucleotides downstream at the 5' end of the next gene in the polycistronic pre-mRNA. Current efforts are directed towards understanding the nature of this connection. We are also studying splice-site recognition in C. elegans, since it appears to be somewhat different from other animals, perhaps because the splicing machinery must cope with both cis-splicing and two kinds of trans-splicing (SL1 and SL2). We have cloned the genes that encode the two subunits of the splicing factor U2AF, which is involved in splice acceptor site recognition. We have discovered that both U2AF subunits interact with the 3' splice site consensus sequence, with the small subunit responsible for recognition of the invariant AG/R at the splice junction. The pre-mRNA that encodes the large subunit of U2AF is alternatively spliced, which may represent a mechanism for autogenous regulation of U2AF levels.