LIN-42, the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription

Abstract

During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression.

Period homolog LIN-42 regulates miRNA transcription to impact developmental timing

“…This was further verified by the use of transcriptional reporters for let-7 and lin-4. 15,16 GFP mRNA and protein levels expressed from these reporters was increased in the absence of lin-42. 15,16 Thus LIN-42 regulates a multitude of miRNAs by inhibiting primary miRNA transcription.…”

“…15,16 GFP mRNA and protein levels expressed from these reporters was increased in the absence of lin-42. 15,16 Thus LIN-42 regulates a multitude of miRNAs by inhibiting primary miRNA transcription. Such regulation is likely carefully spatially and temporally controlled, however, as LIN-42 is expressed in multiple cell types throughout development.…”

“…1 Though a lin-42 mutation suppresses the vulvaless phenotype of lin-4(ma161) worms or the bursting phenotype of let-7(n2853) worms, these phenotypes are not suppressed in lin-4(e912) or let-7 (mn112) worms ( Table 2). [14][15][16] The former alleles reduce, but do not eliminate lin-4 or let-7 expression while the latter alleles are both null. Thus, at a minimum, LIN-42 functions through lin-4 and let-7 miRNAs to impact the heterochronic pathway (Fig.…”

“…Both Van Wynsberghe et al and Perales et al found that levels of mature lin-4 and let-7 are increased in lin-42 mutant worms at multiple stages throughout development. 15,16 This effect of LIN-42 on miRNAs extended far beyond the heterochronic pathway however, as 95% of miRNAs in eggs and »33% of miRNAs in L4 stage worms were upregulated in the absence of lin-42. 15 This increase in mature miRNA levels was accompanied by an increase in pri-let-7 and pri-lin-4 levels.…”

“…Indeed, this regulation has clear effects on cell functions beyond seam cells and the heterochronic pathway. For example, mutations in lin-42 suppress lsy-6 miRNA neuronal cell fate specification phenotypes, 16 likely by increasing lsy-6 levels above a required threshold. Additionally, the finding that LIN-42 also regulates primary miRNA transcription in embryos suggests that LIN-42 also has important functions prior to larval development.…”