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.

sym-1, a leucine-rich repeat-containing gene, is synthetic lethal with mec-8

Andrew G. Davies, Jocelyn E. Shaw, Robert K. Herman

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.