Worm Breeder's Gazette 12(4): 14 (October 1, 1992)
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 previously characterized three Tc1 -inducedmutants of the regulatory myosin light chain gene, mlc-2 (WBG 12(2):59). Each of these mutants contains less than wild-type levels of mlc-2 mRNA. In each case all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. For mlc-2 ( r948 ::T c1 ),the 3' splice acceptor used within Tc1 is unusual; the last dinucleotide of the "intron" is GG rather than the canonical AG typical of eukaryotic introns. To determine if splicing to remove Tc1 from pre-mRNAs is unique to mlc-2 ,or if it is a general property of Tc1 -inducedalleles, we characterized the structures of mRNAs from Tc1 -inducedalleles of unc-54 ,the myosin heavy chain gene and of hlh-1 ,the C. elegans homolog of the vertebrate MyoD gene family (CeMyoD).
We used the PCR in combination with sib-selection (WBG 11(5):65) to isolate Tc1 -inducedmutations of hlh-1 . hlh-1 ( r1010 ::T c1 )is a Tc1 insertion within the hlh-1 second exon. r1010 homozygotes are viable and have no obvious muscle defects. In this mutant, the normal 5' splice donor of the first intron is spliced to a 3' splice acceptor within the right inverted repeat of Tc1 (at position 11). Use of these sites removes all of Tc1 ,except 12 nt, plus 18 nt of hlh-1 from the mature message. The resulting mRNA can be translated in-frame to yield a CeMyoD protein containing an insertion of 4 amino acids from Tc1 ,plus a deletion of 6 amino acids from CeMyoD. The 3' splice acceptor used within Tc1 is unusual; the terminal dinucleotide of this "intron" is TG rather than the canonical AG.
We have also analyzed two Tc1 -inducedalleles of unc-54 that were previously isolated in a genetic screen for muscle defective mutants. These two insertions, r323:: Tc1 and r328:: Tc1 ,are at the same position, but in opposite orientations within the unc-54 third exon. We estimate that these mutants contain 20-50% of wild-type levels of an approximately normally sized unc-54 mRNA. In addition, r328 contains a small amount of an unc-54 mRNA that is approximately 1.6 kb larger than wild-type. In these mutants, a cryptic 5' splice donor, 33 nt upstream of the normal splice donor, is spliced to the normal 3' acceptor of the third intron. Use of these sites removes all of Tc1 plus 33 nt of unc-54 from the mature message. The resulting mRNAs can be translated in-frame to yield myosin heavy chain (MHC) proteins deleted for 11 amino acids. The 5' splice donor within unc-54 is unusual; the first two nucleotides of the "intron" are TT rather that the canonical GT typical of eukaryotic introns. In spite of the small in-frame deletions in the MHC proteins, r323 and r328 are indistinguishable from unc-54 null mutants. The 11 amino acids are deleted from the MHC globular head region; presumably these amino acids are important for MHC protein stability, function or thick filament assembly.
For all six Tc1 -inducedmutations we characterized, a fraction of the mutant pre-mRNA is spliced in a manner that leaves the mature message in-frame. Perhaps a variety of different splice sites are used to splice Tc1 from the mutant pre-mRNAs. We expect mRNAs that are spliced in a manner that leaves nonsense or frameshift mutations in the message would be degraded by the C. elegans smg gene system (WBG 11(5):90). The small amount of in-frame mRNAs present in these mutants is probably stable because these mRNAs are translated throughout their entire length and therefore are not substrates for smg-dependent degradation.