let-7 microRNA,
lin-42 and
kin-20 function in the C. elegans heterochronic pathway to regulate the correct timing of cellular differentiation during larval development.
lin-42 and
kin-20 are homologs of the core circadian timing genes period and casein kinase I epsilon, respectively. Genetic analysis places these two circadian gene homologs downstream of
let-7, but it is not known whether they are directly regulated by
let-7, or whether they constitute a parallel pathway that controls developmental timing. MicroRNAs (miRs) post-transcriptionally downregulate expression of their target genes by attaching to partially complementary sites in the 3'' UTR of the message and preventing translation. We have identified potential
let-7 complementary sites (LCSs) in the 3''UTRs of
lin-42 and
kin-20. In order to determine whether
lin-42 or
kin-20 is post-transcriptionally regulated by
let-7 miR, we used a lacZ reporter gene attached to
lin-42 or
kin-20 3''UTRs.
kin-20 post-transcriptional expression does not appear to be regulated via its 3''UTR. However, we found that two of the three
lin-42 isoforms,
lin-42A and
lin-42B, are down-regulated via their 3''UTRs. This down-regulation during the larval to adult transition is likely to be mediated directly by
let-7 as the regulation is dependent both on the presence of the LCSs and the presence of
let-7 miR. Furthermore, the
let-7 paralogs,
mir-84,
mir-48 and
mir-241, also directly regulate
lin-42A and
lin-42B, but do so at earlier stages of larval development. By using quantitative real time PCR to measure
lin-42 mRNA levels, we found that levels of
lin-42A and
lin-42B are elevated in
let-7 loss-of-function genetic backgrounds. Interestingly, while loss of the
let-7 paralogs in the VT1066 strain resulted in an increase at the L2 stage, subsequent levels of
lin-42A and
lin-42B in L3 and L4 stages were normal, and
lin-42 expression cycling was not disrupted. On the other hand, mRNA levels of
lin-42C, which does not contain any LCSs in its 3''UTR, was unaffected in
let-7 loss-of-function genetic backgrounds compared to N2/wild type. Our results validate
lin-42 as a direct regulatory target of
let-7 family microRNAs, and raise the possibility that
let-7 microRNAs may regulate the circadian timing gene period. In humans, all three period genes have putative LCSs in their 3''UTRs. To test for regulation by
let-7 miRs, we utilized a luciferase reporter gene attached to period 3''UTRs and assayed for reporter expression in human cell lines in which
let-7 expression manipulated. We have found that Hs period 3 is regulated by
let-7 miR.