GLD-1, a KH motif containing RNA binding protein in C.elegans , is a germline specific tumor suppressor that is essential for oocyte development. GLD-1 also functions to promote male sex determination in the hermaphrodite germline and has a redundant function to initiate meiotic development. GLD-1 is abundant in the cytoplasm of early meiotic prophase germ cells in the distal region but absent in developing oocytes in the proximal region. Therefore GLD-1 is thought to regulate the translation and/or the stability of a subset of maternal mRNAs that may have functions in oocyte differentiation, maturation/ovulation and early embryogenesis. We have identified multiple in vivo mRNA targets of GLD-1 by their ability to interact with GLD-1 in cytosol extract. These target mRNAs are preferentially expressed in the germline and as expected, several of them have essential functions in oocyte differentiation, maturation/ovulation and early embryogenesis. Interestingly, subsets of 3 gene families are identified as mRNA targets of GLD-1; chitin binding domain containing, puf -5/-6/-7/-10 and
oma-1/-2 TIS11 zinc finger containing. In each subset, single RNAi has no detectable phenotype while double RNAi results in a germline or early embryonic phenotype, suggesting that GLD-1 binds and co-regulates functionally redundant homologs. Analysis of three mRNA targets (
rme-2,
oma-1 and
oma-2 ), reveals that GLD-1 acts as a translational repressor. Antibody staining of wild-type hermaphrodite germline shows that the corresponding proteins for three mRNA targets are absent from the distal region where GLD-1 is abundant, while they increase in abundance in growing oocytes in the proximal region where GLD-1 levels fall precipitously. Consistent with GLD-1 functioning as a translational repressor, they prematurely accumulate in the distal region of
gld-1 null hermaphrodites. These data imply that GLD-1 is likely acting as a translational repressor for most mRNA targets. However, two mRNA targets, including T23G11.2, are unstable in
gld-1 null animals, suggesting that GLD-1 binds and stabilizes them. Upon careful inspection, T23G11.2 mRNA has two small upstream open reading frames in 5’-UTR and therefore is likely a target of nonsense mediated mRNA decay (NMD). In situ staining shows that the level of T23G11.2 mRNA is high in the distal region of wild-type but is very low in developing oocytes in the proximal region. In contrast, T23G11.2 mRNA accumulates to high levels in developing oocytes of
smg-2 mutants, which lack NMD. In addition, T23G11.2 mRNA is undetectable in
gld-1 null germlines but is present in
gld-1 null
smg-2 double mutant germlines. GLD-1 thus functions to bind and protect T23G11.2 mRNA from NMD, presumably by repressing translation. At least four GLD-1 binding sites have been identified so far in three mRNA targets. They are located in 5’-UTR, 5’-end of ORF, and 3-UTR, suggesting that GLD-1 can bind to 5’-end, 3’-end, or both ends depending on the mRNA. For
rme-2 mRNA, GLD-1 binds specifically to both the 5’- and the 3’-ends. While a missense mutation (
q361 ) in the invariant GXXG residues inside the KH motif completely abolishes the RNA binding activity of GLD-1, another missense mutation (
q126 ) in the conserved C-terminal region flanking the KH motif only affects binding to the previously identified target mRNA
tra-2 (Jan et al ., 1999). This suggests that GLD-1 could have more than one binding specificity or binding could be modified by the formation of complexes with other RNA binding proteins, which could allow GLD-1 to control diverse mRNAs.