Worm Breeder's Gazette 10(2): 11
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 characterizing the C. elegans small nuclear RNAs ( snRNAs) and their genes (see Thomas, Zucker-Aprison and Blumenthal, this issue). snRNAs are the RNA component of small nuclear ribonucleoproteins (snRNPs) which catalyze various nuclear RNA processing events. The snRNPs which we have been studying, U1, U2, U4, U5 and U6, have been shown to catalyze cis-splicing of mRNA introns in human cells and in yeast. As probes we have used DNA oligomers designed to hybridize to highly conserved sequences in the snRNAs. In other organisms the snRNAs U1, U2 U4, and U5 have been shown to be bound to the Sm protein and have a special 2,2,7-methyl-guanosine ( trimethyl-guanosine, TMG) cap. The U6 snRNA lacks these features, however it is generally present in anti-Sm or anti-TMG cap immunoprecipitates via its ability to associate with the U4 snRNA by intermolecular base-pairing. We immunoprecipitated C. elegans nuclear extracts with anti-Sm and anti-TMG cap antibodies, extracted the RNAs, separated them on denaturing polyacrylamide gels, and blotted them onto nylon. When these blots were probed with [32P]- labeled oligonucleotides, we found U1, U2, U4, U5 and U6 snRNAs present in the immunoprecipitates, as expected. We also immunoprecipitated snRNAs from deproteinized total C. elegans RNA with anti-TMG cap antibody and analyzed them in the same way. We found U1, U2, U4, U5 and a small amount of U6 snRNAs in these immunoprecipitates. However, if we heated and quick-cooled the total RNA before the immunoprecipitation, there was no U6 snRNA, consistent with the idea that U6 and U4 interact by base pairing. Surprisingly, when we probed these same blots with a labeled DNA oligomer complementary to the trans-spliced leader RNA, we found the leader RNA present in all of the immunoprecipitates. The leader RNA's presence in the anti-Sm immunoprecipitates suggests strongly that it interacts with the Sm protein. The existence of a consensus Sm- binding site in the leader RNA's 'intron' portion suggests that this interaction is direct. The immunoprecipitability of the leader RNA with anti-TMG cap antibody was insensitive to heating and quick- cooling, hence it must interact directly with this antibody. To prove that this interaction was not due to a cross-specificity of the antibody, we showed that the compound 7-methyl GpppG (the standard mRNA cap) competes with the leader RNA for binding to the antibody as inefficiently as it competes with U1, U2, U4 and U5 snRNAs. We conclude that the leader RNA has properties of an snRNA. If the leader RNA snRNP is indeed a substrate for transsplicing, then one would predict that a 'Y'-structure product (analogous to the lariat of cis-splicing) would be Sm-bound and that the recipient mRNAs would have 2,2,7-methylguanosine caps at their 5'-ends. We have tested the latter prediction by analyzing the supernatant and pellet from an anti-TMG cap immunoprecipitation of total C. elegans RNA on blots of formaldehyde-agarose gels. While the U2 snRNA and the leader RNA were present in the pellet, actin mRNAs were present only in the supernatant. Furthermore, we saw diffuse hybridization to an oligonucleotide complementary to the 'exon' portion of the leader RNA only in the supernatant, which we interpret as representing the sum of the trans-spliced RNAs. These data indicate that the mature RNAs that have been trans-spliced lack the TMG cap structure of the leader RNA. This result suggests the existence of a cap modifying or replacing activity whose substrate is mRNAs that have been trans-spliced. Our findings have important implications for the mechanism of trans- splicing in C. elegans. The leader RNA, discovered as substrate of trans-splicing, shares properties with snRNPs, which were heretofore known as catalysts of RNA splicing. Since the splice sites used in trans-splicing fit the consensus for splice sites used in cis-splicing, it seems likely that the two types of splicing are mechanistically related. That the leader RNA has properties of an snRNP suggests it may have special interactions with other snRNPs that catalyze its joining to recipient RNAs in C. elegans.