Mature microRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. Though miRNAs are essential for many fundamental cell processes, the mechanisms regulating miRNA biogenesis are poorly understood. miRNA biogenesis is a multi-step process in which long, genetically-encoded primary (pri-) miRNAs are cleaved into precursor (pre-) and then mature miRNAs. The
let-7 miRNA is conserved across phyla, an important tumorigenesis regulator in humans and crucial for proper development in C. elegans.
Pri-let-7 is transcribed from two transcriptional start sites and trans-splicing to the Spliced Leader 1 RNA produces a third isoform important for mature
let-7 production. Unexpectedly, by reporter and molecular analyses we found that pri- and mature
let-7 expression is uncoupled suggesting that pri-
let-7 is post-transcriptionally regulated. Since mutations in the RNA binding protein LIN-28 or the period protein homolog LIN-42 suppress strong loss-of-function
let-7 alleles, we investigated the mechanisms by which these heterochronic pathway members might regulate
let-7 biogenesis. Recently, distinct models by which LIN-28 controls mammalian
let-7 miRNA expression were proposed. To ascertain if C. elegans LIN-28 similarly regulates
let-7 expression, we studied
let-7 biogenesis in
lin-28(
n719) worms. Contrary to WT worms, we find that concurrent with initial pri-
let-7 expression, pre- and mature
let-7 accumulate prematurely during late L1 in
lin-28 mutants. Interestingly, pri-
let-7 isoform levels vary in
lin-28(
n719) worms pointing to a specific pri-
let-7 isoform as the target of LIN-28 repression. Surprisingly,
lin-28 also differentially regulates maturation of
let-7 sister miRNAs, suggesting that
lin-28 activity can positively and negatively affect miRNA biogenesis. Unlike LIN-28, which affects specific miRNAs, we found that LIN-42 globally regulates miRNA expression. Using a previously identified allele and a novel C terminal allele of
lin-42, which was identified as a spontaneous
let-7 suppressor, we discovered that LIN-42 activity down-regulates mature
let-7 levels. Our results suggest that LIN-42 post-transcriptionally regulates
let-7 expression since pri-
let-7 levels are unchanged, pre-
let-7 levels decrease and mature
let-7 levels increase in
lin-42 mutant worms. We observed similar effects on pre- and mature miRNA levels for other embryonic and larval stage miRNAs in
lin-42 mutant worms. Thus, we hypothesize that LIN-42 inhibits pre to mature miRNA processing. Our results propose a new role for the period protein homolog in globally regulating miRNA processing in C. elegans and raise the possibility that period proteins in other organisms could play similar roles.