Developmental timing in C. elegans is regulated by the heterochronic pathway, a conserved developmental program that features temporal waves of miRNA expression. This mechanism coordinates multiple stage-specific developmental events, including the late-larval morphogenesis that generates the rounded adult male tail tip. We identified a novel gene, lep-5 (H36L18.2), that regulates the timing of tail tip morphogenesis in L4 males. Loss of lep-5 results in absent or delayed morphogenesis, such that adult males retain a juvenile pointed tail tip, while overexpression of lep-5 causes premature retraction. Some mutant adults undergo an additional molt, thus demonstating that the activity of lep-5 is not restricted to the tail tip. However, lep-5 mutants do not exhibit any defects in the development of the lateral seam. Unexpectedly, lep-5 functions not as a protein or miRNA, but rather as a novel long noncoding RNA (lncRNA). SL1-spliced lep-5 lncRNA is predicted to adopt a complex secondary structure with multiple stem-loops, at least two of which are necessary for its function. lep-5 is highly conserved within Caenorhabditis but could not be identified outside of this genus. The expression of lep-5 is temporally regulated with a peak between late L2 and mid L3. During this time, lep-5 lncRNA is visible via RNA smFISH in many tissues except the gut and germline, with a notable concentration in the nervous system.
By testing genetic interactions and performing Western blot and qPCR analysis, we determined that lep-5 lncRNA down-regulates LIN-28 protein levels post-transcriptionally without affecting LIN-28 mRNA levels. We further found that Lep-5 acts on the 3’UTR of the LIN-28 mRNA independent of previously identifid miRNA binding sites. Although the mechanism for its activity is not yet known, lep-5 represents the first example of a functional lncRNA in C. elegans. Furthermore, adding a lncRNA to the multiple miRNAs that act in the heterochronic pathway highlights the striking importance of RNA-mediated regulation in developmental timing.
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