Cold Shock Domain Protein LIN-66 cooperates with microRNA-pathway buffering to safeguard developmental timing.

Robust execution of developmental cell fates requires precise spatiotemporal control of the fate-defining regulators. In Caenorhabditis elegans , temporal patterning of larval hypodermal fates is governed by the heterochronic gene regulatory network, in which microRNAs act as major post-transcriptional regulators by silencing temporal transcripts through 3 ' UTR-dependent repression. Here, we investigate lin-66 , which encodes a nematode-specific cold shock domain protein previously implicated in heterochronic regulation and reported to associate with the miRISC effector protein AIN-1. Using targeted domain mutations and genetic analysis, we show that LIN-66 activity in the hypodermal cell-fate patterning requires its cold shock domain. Loss of lin-66 causes persistent expression of LIN-14 and LIN-28, two early temporal regulators in the hypodermal seam cells that are canonical microRNA targets. Analysis indicates that lin-66 function in seam-cell fate patterning does not depend on the native 3 ' UTR sequences of lin-14 or lin-28 , distinguishing its activity from canonical microRNA repression.. Consistent However, consistent with the a broad functional overlap between LIN-66 function and microRNA-mediated regulation, hypodermal lin-66 loss-of-function phenotypes are strongly enhanced by mutations in alg-1 and ain-1/2 , which encode components of the microRNA-induced silencing complex. Moreover, loss of lin-66 enhances phenotypes in mutants sensitized for microRNA activity outside the hypodermis. Together, these findings identify LIN-66 as a cold shock domain-dependent post-transcriptional regulator that safeguards developmental timing by limiting persistence of early fate regulators through mechanisms that intersect with, but are partly separable from, canonical 3 ' UTR-mediated microRNA repression.