Worm Breeder's Gazette 15(1): 79 (October 1, 1997)
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.
Dept. of Genetics and Cell Biology University of Minnesota 1445 Gortner Ave. St. Paul, MN 55108 U.S.A.
The mec-8 gene encodes a protein with two RNA recognition motifs (RRMs) and affects the accumulation of certain alternatively spliced transcripts of unc-52 (Lundquist et al., 1996 Development 122: 1601). The pleiotropic phenotype of mec-8 mutants suggests that MEC-8 affects the processing of RNA from multiple target genes. Previously, Spike and Herman (1997 WBG 14(5): 61) described the identification of a mutation, mn601, that is synthetic lethal with a mec-8 mutation. mn601 has been assigned to the gene sym-1 (synthetic lethal with mec-8). Animals homozygous for sym-1 alone are essentially wild type, but mec-8; sym-1 double mutants (bearing any of several mec-8 mutations we have tested) arrest development embryonically. We can imagine three mechanisms by which a mutation could be synthetic lethal with a mec-8 mutation. First, as is the case with certain alleles of unc-52, the synthetic lethal mutation could occur in a target gene with alternatively processed transcripts where at least one class of transcripts is required for viability; mec-8 mutation would affect certain transcripts, and the synthetic lethal mutation would affect others. Second, the synthetic lethal mutation could be in a gene that provides an RNA processing function overlapping with that of MEC-8 such that the products of mec-8(+) and sym-1(+) would each be capable of processing the transcripts of an essential target gene. Third, the synthetic lethal mutation could occur in a gene that provides a function overlapping with that of a gene whose transcripts are processed by MEC-8; in this case, a mutation in mec-8 would lead to the loss of the target gene function, which would make the otherwise-redundant gene essential. These alternatives lead to different molecular consequences. In the first mechanism, the mec-8 target gene should exhibit mec-8-dependent processing of a subclass of its transcripts. The second mechanism implicates a protein involved in RNA processing. The third mechanism makes no predictions about function except that it is limited to the range of functions possessed by targets of mec-8. sym-1 was mapped 1.0 map unit left of unc-3. We injected DNA from YACs and cosmids from this region and found that Y52C11, Y52F5, Y39H3, C44H4 and T01F4 (all of which overlap) rescued the temperature-sensitive synthetic lethal phenotype of mec-8(u218ts); sym-1(mn601) animals. We are grateful that the sequence of C44H4 has been determined by the Genome Sequencing Consortium. A ~15kb PCR fragment derived from C44H4 rescued and is predicted, by Genefinder and analysis of cDNAs, to contain three transcription units, C44H4.3-C44H4.5. A 4.4-kb BamHI/PstI fragment containing C44H4.3 provided rescue, while a clone containing a frameshift introduced early in the predicted open reading frame of C44H4.3 failed to rescue the sym-1 mutation. The C44H4.3 gene from the sym-1(mn601) strain was sequenced entirely and a single point mutation resulting in a premature stop codon was found; the mutant gene would produce a protein about two-fifths the wild-type length. The sym-1 gene is predicted to encode a protein with 15 contiguous leucine-rich repeats (LRRs, each about 24 amino acid residues in length) and a threonine/glutamic acid-rich carboxyl terminus. LRRs have been implicated in protein-protein interactions in a broad range of proteins including adhesion molecules and binding partners of RNA-binding proteins. Although some LRR-containing proteins have been implicated in RNA processing events, the diverse functions of other LRR proteins means that we cannot yet rule out any of the mechanisms for synthetic lethality with mec-8 described above. We are testing transcripts of sym-1 for mec-8-dependent alternative processing. In addition, we are constructing a sym-1::gfp translational fusion, the tissue and cellular localization of which may provide insight into a function for sym-1. It is interesting that the two genes immediately upstream of sym-1, C44H4.1 and C44H4.2, are also predicted to encode LRR-containing proteins with similarity to sym-1. We plan to investigate the roles of these genes using antisense-RNA injections.