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Lin28/LIN-28 is a conserved RNA-binding protein that promotes proliferation and pluripotency and can be oncogenic in mammals. Mammalian Lin28 and C. elegans LIN-28 have been shown to inhibit biogenesis of the conserved cellular differentiation-promoting microRNA let-7 by directly binding to unprocessed let-7 transcripts. Lin28/LIN-28 also bind and regulate many mRNAs in diverse cell types. However, the determinants and consequences of LIN-28-mRNA interactions are not well understood. Here, we report that C. elegans LIN-28 represses the expression of LIN-46, a downstream protein in the heterochronic pathway. We find that lin-28 and sequences within the lin-46 5' UTR are required to prevent LIN-46 expression at early larval stages. Moreover, we find that precocious LIN-46 expression caused by mutations in the lin-46 5' UTR is sufficient to cause precocious heterochronic defects similar to those of lin-28(lf) animals. Thus, our findings demonstrate the biological importance of the regulation of individual target mRNAs by LIN-28., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

MicroRNAs target complementary mRNAs for degradation or translational repression, reducing or preventing protein synthesis. In Caenorhabditis elegans , the transcription factor HBL-1 (Hunchback-like 1) promotes early larval (L2)-stage cell fates, and the let-7 family microRNAs temporally downregulate HBL-1 to enable the L2-to-L3 cell-fate progression. In parallel to let-7 -family microRNAs, the conserved RNA-binding protein LIN-28 and its downstream gene lin-46 also act upstream of HBL-1 in regulating the L2-to-L3 cell-fate progression. The molecular function of LIN-46, and how the lin-28-lin-46 pathway regulates HBL-1, are not understood. Here, we report that the regulation of HBL-1 by the lin-28-lin-46 pathway is independent of the let-7 / lin-4 microRNA complementary sites (LCSs) in the hbl-1 3'UTR, and involves stage-specific post-translational regulation of HBL-1 nuclear accumulation. We find that LIN-46 is necessary and sufficient to prevent nuclear accumulation of HBL-1. Our results illuminate that robust progression from L2 to L3 cell fates depends on the combination of two distinct modes of HBL-1 downregulation: decreased synthesis of HBL-1 via let-7 -family microRNA activity, and decreased nuclear accumulation of HBL-1 via action of the lin-28 - lin-46 pathway., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Like mammalian neurons, Caenorhabditis elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in anterior ventral microtubule (AVM) axon regeneration. In older AVM axons, let-7 inhibits regeneration by down-regulating LIN-41, an important AVM axon regeneration-promoting factor. Whereas let-7 inhibits lin-41 expression in older neurons through the lin-41 3' untranslated region, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures that axon regeneration is inhibited only in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously shown to control timing of events in mitotic stem cell lineages, is reutilized in postmitotic neurons to control postdifferentiation events.

Animals developing in the wild encounter a range of environmental conditions, and so developmental mechanisms have evolved that can accommodate different environmental contingencies. Harsh environmental conditions cause Caenorhabditis elegans larvae to arrest as stress-resistant "dauer" larvae after the second larval stage (L2), thereby indefinitely postponing L3 cell fates. HBL-1 is a key transcriptional regulator of L2 vs. L3 cell fate. Through the analysis of genetic interactions between mutations of hbl-1 and of genes encoding regulators of dauer larva formation, we find that hbl-1 can also modulate the dauer formation decision in a complex manner. We propose that dynamic interactions between genes that regulate stage-specific cell fate decisions and those that regulate dauer formation promote the robustness of developmental outcomes to changing environmental conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally via antisense base-pairing. Although miRNAs are involved in a variety of important biological functions, little is known about their transcriptional regulation. Using yeast one-hybrid assays, we identified transcription factors with a FLYWCH Zn-finger DNA-binding domain that bind to the promoters of several Caenorhabditis elegans miRNA genes. The products of the flh-1 and flh-2 genes function redundantly to repress embryonic expression of lin-4, mir-48, and mir-241, miRNA genes that are normally expressed only post-embryonically. Although single mutations in either flh-1 or flh-2 genes result in a viable phenotype, double mutation of flh-1 and flh-2 results in early larval lethality and an enhanced derepression of their target miRNAs in embryos. Double mutations in flh-2 and a third FLYWCH Zn-finger-containing transcription factor, flh-3, also result in enhanced precocious expression of target miRNAs. Mutations of lin-4 or mir-48&mir-241 do not rescue the lethal flh-1; flh-2 double-mutant phenotype, suggesting that the inviability is not solely the result of precocious expression of these miRNAs. Therefore, the FLH-1 and FLH-2 proteins likely play a more general role in regulating gene expression in embryos.

A temporal gradient of the novel nuclear protein LIN-14 specifies the timing and sequence of stage-specific developmental events in Caenorhabditis elegans. The profound effects of lin-14 mutations on worm development suggest that LIN-14 directly or indirectly regulates stage-specific gene expression. We show that LIN-14 can associate with chromatin in vivo and has in vitro DNA binding activity. A bacterially expressed C-terminal domain of LIN-14 was used to select DNA sequences that contain a putative consensus binding site from a pool of randomized double-stranded oligonucleotides. To identify candidates for genes directly regulated by lin-14, we employed DNA microarray hybridization to compare the mRNA abundance of C. elegans genes in wild-type animals to that in mutants with reduced or elevated lin-14 activity. Five of the candidate LIN-14 target genes identified by microarrays, including the insulin/insulin-like growth factor family gene ins-33, contain putative LIN-14 consensus sites in their upstream DNA sequences. Genetic analysis indicates that the developmental regulation of ins-33 mRNA involves the stage-specific repression of ins-33 transcription by LIN-14 via sequence-specific DNA binding. These results reinforce the conclusion that lin-14 encodes a novel class of transcription factor.

Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. We suggest that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29.

A hierarchy of heterochronic genes, lin-4, lin-14, lin-28 and lin-29, temporally restricts terminal differentiation of Caenorhabditis elegans hypodermal seam cells to the final molt. This terminal differentiation event involves cell cycle exit, cell fusion and the differential regulation of genes expressed in the larval versus adult hypodermis. lin-29 is the most downstream gene in the developmental timing pathway and thus it is the most direct known regulator of these diverse processes. We show that lin-29 encodes a protein with five zinc fingers of the (Cys)2-(His)2 class and thus likely controls these processes by regulating transcription in a stage-specific manner. Consistent with this role, a lin-29 fusion protein binds in vitro to the 5' regulatory sequences necessary in vivo for expression of col-19, a collagen gene expressed in the adult hypodermis. lin-29 mRNA is detected in the first larval stage and increases in abundance through subsequent larval stages until the final molt, when lin-29 activity is required for terminal differentiation.