Worm Breeder's Gazette 10(1): 66
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.
There are several reasons for expecting mechanistic aspects of splicing to be particularly interesting in C. elegans: 1. The introns are unusually short, generally about 50 bases; 2. They have an unusually highly conserved 3' boundary consensus sequence, TTTCAG, and no polypyrimidine stretch just upstream of the 3' intron boundary; 3. There is no obvious consensus sequence for a branch site; and 4. C. elegans has both cis- and trans-splicing. Hence, we have begun studying the nematode splicing machinery. In other eukaryotes it has been shown that the splicing machinery consists of a set of RNA protein complexes called snRNPs. The U1 snRNP interacts with the 5' splice site, the U2 snRNP with the branch site, and the U5 snRNP probably interacts with the 3' splice site. U4 and U6 snRNPs are also required but their roles are less well defined. Our approach has been to use DNA oligomers designed to hybridize to conserved regions of the RNA components of snRNPs - the snRNAs. When we probe blots of total C. elegans RNA with these oligomers, we see bands of the expected mobilities corresponding to all of the snRNAs implicated in splicing. On blots from 10% acrylamide, 7M urea gels we see multiple (4 to 6) bands that hybridize to the U1 and U2 oligomers, while only a single band hybridizes to these oligomers in yeast and fly RNA control lanes. However, on blots from more denaturing 15% acrylamide, 99% formamide gels there is only a single band of hybridization to the U1 oligomer and two bands of hybridization to the U2 oligomer. Interestingly, the U2 oligomer is designed to hybridize to the region of U2 RNA which has been shown to interact with the branch site by base pairing in yeast. Thus it appears that this region of C. elegans U2 is conserved despite the poor sequence conservation at the C. elegans intron branch sites. We have also used these oligomers as primers for limited RNA sequencing of U2, U4 and U6 RNAs. These worm snRNAs are quite different in primary sequence from the mammalian and yeast snRNAs. Our preliminary data indicate, however, that worm U2 can fold into a secondary structure which has been conserved from yeast to man. We have begun cloning the genes encoding the C. elegans snRNAs using these same oligomers. To date we have six different lambda clones which hybridize to the U2 oligomer. Three of these have two regions of hybridization, consistent with there being at least nine U2 genes. A genomic Southern probed with a DNA fragment containing one of these U2 genes (confirmed by sequencing) shows approximately eleven bands. Thus it appears that in C. elegans there is a family of U2 genes of ten to twelve members.