In C. elegans, the miRNAs
lin-4 and
let-7 control certain stage-specific developmental programs. In
lin-4 mutants, early larval programs are reiterated throughout development. Similarly,
let-7 is required late in larval development to direct terminal differentiation of epithelial cells and exit from the molting cycle. Both block the translation of target genes by binding to imperfectly complementary sites in the target mRNAs, like most animal miRNAs studied to date. We have explored the function of one
let-7 paralog,
mir-84, through a combination of genetics and overexpression studies.Loss of
mir-84 by chromosomal deletion greatly enhanced the penetrance of entry into a supernumerary molt caused by the weak
let-7(
mg279) mutation. Consistent with this,
let-7(
mg279)
mir-84(
tm1304) adults expressed two GFP reporters for molting (
mlt-10p::GFP-PEST and
nas-37p::GFP-PEST) that wild-type animals express only before larval molts. By 20 hours of adulthood, 99% of
let-7(
mg279)
mir-84(
tm1304) animals expressed
mlt-10p::GFP-PEST. Some
let-7(
mg279) single mutant adults also expressed
mlt-10p::GFP-PEST, but only later in adulthood. Inactivation of the precocious heterochronic genes
lin-14,
lin-28,
lin-42,
lin-41 and
hbl-1 strongly suppressed supernumerary molting and expression of
mlt-10p::GFP-PEST in
let-7(
mg279)
mir-84(
tm1304) adults. This suggests that
mir-84 and
let-7 act through targets in the heterochronic pathway to direct cessation of molting.Because
mir-84 might function redundantly with other miRNAs, masking phenotypes resulting from the loss of a single gene, we further explored
mir-84 function through overexpression.
mir-84 overexpression caused precocious vulval development and differentiation of the seam cells, phenotypes reminiscent of
lin-14 and
lin-28 mutants.
mir-84 overexpression also suppressed
lin-4 and
let-7 loss-of-function phenotypes, suggesting that
mir-84 functions partly redundantly with both of these miRNAs.
mir-84 is complementary to at least one site in the
lin-14 3 UTR, and
mir-84 overexpression caused precocious down-regulation of both LIN-14 and a
col-10p::lacZ::
lin-14 3 UTR reporter. These results suggest that
mir-84 acts together with
lin-4 to regulate
lin-14 through its 3 UTR, consistent with the fact that
mir-84 expression begins in the L1 stage. A
mir-84::GFP reporter was expressed in a temporally regulated way in the somatic gonad (including the anchor cell), the vulval precursors, the vulva, the pharynx and the seam cells, consistent with the phenotypes observed when
mir-84 was deleted or overexpressed.