Worm Breeder's Gazette 16(5): 44 (February 1, 2001)
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.
Department of Genetics, Cell Biology, and Development, University of Minnesota. St. Paul, MN 55108
Mutations in smu-2 were isolated as extragenic suppressors of the synthetic lethal phenotype of mec-8 unc-52(viable) double mutants. The smu-2 mutations also suppress other phenes of mec-8 mutants, such as mechanosensory and chemosensory defects, apparently by a bypass mechanism; mec-8 encodes a putative RNA binding protein that affects the accumulation of certain alternatively-spliced transcripts of unc-52 and other genes (Lundquist et al. Development 1996 122: 1601-1610). Finally, smu-2 mutations suppress the uncoordination conferred by nonsense mutations in exon 17 but not exon 18 of unc-52. We hypothesized that smu-2 encodes a factor that regulates the splicing of various target genes, at least some of which are also targets of splicing control by MEC-8. Indeed, our RT-PCR experiments indicate that mutation in smu-2 leads to enhanced skipping of exon 17 but not exon 18 of unc-52 pre-mRNA.
We identified smu-2 by positional cloning and transformation rescue. We rescued the smu-2 mutant phenotype (recessive suppression of unc-52 and mec-8) with the YAC Y37F3 and with a long-range PCR product that contained the single gene Y49F6B.4. We also found that a construct containing 2 kb of the promoter region of Y49F6B.4 inserted 5' to a full length cDNA (YK563h8) rescued smu-2(mn416) and that a frameshift mutation introduced in the same construct abolished rescue. Finally, we identified sequence alterations in Y49F6B.4 associated with all three known smu-2 mutations.
Database searches showed that smu-2 is 37% identical to a mammalian nuclear protein called RED. The similarities between these proteins occur throughout their full extents. SMU-2 is the only protein with significant similarity to RED in the C. elegans genomic sequence database. Neubauer et al. (Nat. Genet. 1998 20: 46-50) identified RED in purified human splicesomes using mass spectrometry and peptide sequencing. The stringent conditions under which the spliceosomes were isolated--proteins transiently associated with spliceosomes were not isolated--suggest that RED is tightly associated with the spliceosome. RED was named after the most distinctive feature of the protein, a domain consisting of alternating basic (arginine) and acidic residues (aspartic and glutamic acid). RED domains are similar to RD domains that consist mainly of arginine and glutamic acid di-peptide repeats. RD domains have been found in a number of proteins that associate with the spliceosome. The human RED domain has diverged from SMU-2's RED domain--SMU-2 contains far more RD di-peptide repeats, and in addition it contains five serine residues. Surprisingly, Drosophila and Arabidopsis homologues do not contain a RED domain at all, although there is high conservation in regions outside this domain.
None of our three smu-2 mutations is a molecular null. mn416 is a 3' splice site mutation, mn610 is a single base pair deletion in the last quarter of the protein, and mn611 is a rearrangement with a breakpoint in the last quarter of the protein; thus, both mn610 and mn611 are predicted to make the N-terminal three-quarters of the protein, including the RED domain. We gained further evidence that these mutations are not null when we did transformation rescue experiments using long-range PCR products generated from mutant DNA templates: mutant DNA was able to rescue the smu-2 phenotype, presumably as a consequence of overexpression. We did RNAi experiments to see if we could detect a more severe smu-2 phenotype. When we injected smu-2 dsRNA (including the RED domain) into unc-52(ts) mutants, we mimicked the smu-2 mutant phenotype: over 98% suppression of unc-52 and no embryonic lethality.
We have monitored expression of a rescuing smu-2::gfp reporter construct: the SMU-2::GFP is a nuclear protein that is ubiquitously expressed at all stages of development.