Worm Breeder's Gazette 11(2): 20b
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.
In a previous gazette (Opus 10#2), we reported that high copy numbers of constructs designed to express minus strand RNA for the unc- 22 and unc-54 loci can cause phenotypes similar to loss of function of the respective genes. These phenotypes were accompanied by a specific decrease in the corresponding proteins. We proposed that the decrease in expression was an antisense RNA phenomenon but could not at that point prove that assertion. We have now convinced ourselves that this phenomenon is due to antisense disruption of gene expression. To date, two different segments of unc-54 gene and three segments of unc-22 have been expressed as minus strand RNA and each gives rise to the expected phenotype. The muscle expression vectors used do not give any phenotypes by themselves. This argues against a specific effect of a single region of one or both of the genes. To confirm that the disruption effect requires homology between the 'antisense' and 'sense' RNAs, we used an in frame deletion unc-22(sb528), which lacks a 1964bp region of the unc-22 gene but has nearly normal unc-22 function (Kiff et. al. Nature 331, 651). A 1427 bp fragment contained entirely within this 1964bp fragment was cloned in antisense orientation into a muscle expression vector to make plasmid pPD34.147. When injected into N2, this plasmid yields many twitchers, while no twitchers have been derived after many injections into unc-22(sb28).To analyze RNA structures and levels, we have taken advantage of one strain (PD68) in which a long tandem array containing the unc-22 antisense plasmid pPD10.46 has integrated, leading to a stable strong twitcher phenotype. Both PCR and quantitative RNAse protections were used to characterize the RNA species produced. To our surprise we found the antisense RNA present at high levels in PD68: about 20 fold more abundant than the endogenous unc-22 message. Transcripts from the endogenous unc-22 locus are also present in PD68, with no evident decrease in abundance over wild type. Antisense construct pPD10.46 contains unc-54 introns 1 to 4, and our analysis has demonstrated that intron 4 is properly and quantitatively spliced. Similarly, an unc-22 intron present in the antisensed region is properly and quantitatively spliced from the endogenous unc-22 transcript in PD68. Thus it appears that the presence of excess cRNA is not interfering with splicing of the endogenous gene. Bass and Weintraub (Cell 55, 1089) describe an unwinding activity from Xenopus that covalently modifies ds RNA, efficiently converting adenines to inosines. If such an activity were active in C. elegans muscle then A to G transitions would be expected in the PCR products derived from the RNAs (see Kimmelman and Kirschner Cell 59, 687). We have cloned and partially sequenced several independent sense and antisense cDNAs (>2kb of total sequence examined) and not found any evidence for the expected transitions. The presence of the spliced transcript of the unc-22 gene at normal levels as well as the lack of the covalent unwinding activity suggest that the antisense RNA interferes with expression by interfering with a late RNA processing or transport step, or (perhaps most likely) by interfering with translation. As a pilot for disruptions of essential genes, we have injected an antisense construct with a segment of the essential gene myo-3 inverted in a muscle expression vector. Several transformed lines were obtained. These lines have a high copy number of the injected DNA present as an extrachromosomal array, and an uncoordinated phenotype which appears mosaic. The lines segregate some dead embryos with a phenotype similar to that seen with myo-3 null mutations ( Waterston, EMBO J 8, 3429). These results are consistent with observation in animals carrying unc-22 and unc-54 antisense constructs of a range of disrupted phenotypes, varying between subtle loss of function to almost complete knockout of the genes.