glp-4 is defined by the mutation
bn2, which at a restrictive temperature (25C) results in adult hermaphrodites that are apparently normal somatically but with few germ cells.
glp-4(
bn2) has been widely used to generate germline-deficient animals for studies of aging, pathogenesis, stress resistance and to assess germline versus somatic gene expression.
To identify the encoded gene product,
glp-4(
bn2) was sequenced to >30x coverage using the Illumina Genome Analyzer. Within the ~2Mb genetically mapped region containing
bn2, coding changes were identified in 5 genes. RNAi of these genes identified only one that gave a sterile phenotype, Y87G2A.5 (
vars-2).
glp-4(
bn2) failed to complement the
vars-2 deletion
tm3947 for fertility at 25C. Tightly linked suppressors (
bn2bn39,
bn2bn40 &
bn2oz283) were isolated and sequencing identified missense mutations within
vars-2.
vars-2 encodes a cytoplasmic class I valyl amino-acyl tRNA synthetase that catalyzes attachment of valine to its cognate tRNA.
bn2 Gly296Asp and the intragenic revertants are in different parts of the CP-1 editing domain. Homology modeling of VARS-2 with the crystal structure of valRS from T. thermophilus suggests a molecular mechanism for the
bn2 mutation (inappropriate deacylation) and suppression by the revertants. The sterile phenotype of
glp-4(
bn2) at 25C likely results from a reduced pool of charged valyl-tRNA leading to reduced protein synthesis.
vars-2 likely also functions in the soma: 1)
vars-2(
tm3947) is larval lethal; 2) in situ hybridization shows somatic expression; and 3) structural analysis suggests that
vars-2 has unique functions that can not be compensated by paralog
vars-1 - VARS-2 is predicted to charge tRNAs with all four Val anti-codons while VARS-1, which has a bulky Trp residue in the anti-codon recognition pocket, is likely unable to bind anti-codons with third position purines. Thus
glp-4(
bn2) may also result in a reduction in protein synthesis in somatic tissues even though there is no obvious somatic phenotype; this occurs with the
rrt-1(
gc47) mutation in arginyl-amino-acyl tRNA synthetase, which results in reduced protein synthesis yet does not display strong somatic phenotypes. Since reduced translation can lead to alterations in lifespan and stress resistance, this property, rather than germline depletion, may be the cause of the
glp-4(
bn2) organismal aging and stress resistance phenotypes.