The mRNA silencing dual activity of the RNA-binding protein LIN41 and the role of its targets in C. elegans development

The microRNA let-7 is an ancient and fundamental regulator of stem cell self-renewal and differentiation in animals. In Caenorhabditis elegans (C. elegans), where let-7 was discovered, it controls self-renewal of stem-cell like cells in the worm skin, the seam cells. In this work, we find that let-7 controls seam cell self-renewal exclusively through one key target, the RNA-binding protein LIN41. Interestingly, LIN41 is a conserved let-7 target, and was shown to regulate developmental switches from a self-renewal to a differentiation program in mouse and human embryonic stem cells, as well as in mouse neural progenitor cells. Despite its prominent role in stem cell development, the physiologically relevant mRNA targets of LIN41, to which it binds to regulate stem cell development, have been unknown in both mammals and C. elegans. LIN41 is reported to repress target mRNAs by inducing mRNA degradation, but has also been speculated to be a translational repressor. In this work, we establish and optimize the ribosome profiling technique for C. elegans, to subsequently study gene expression changes at the level of mRNA abundance and translation, in mutant worms with abnormally high LIN41 levels. With these experiments, performed in vivo in a whole organism, we identify candidate physiological LIN41 target mRNAs. Of those, we confirm four as direct LIN41 targets, as they are enriched in RNA co-immunoprecipitations of LIN41. Surprisingly, the repression mechanisms differ among the four targets, with LIN41 destabilizing mab-10, mab-3, and dmd-3 transcripts, but translationally repressing the lin-29A mRNA. Consistent with its few reported mammalian targets, LIN41 silences three targets by binding to their 3’UTRs, but unexpectedly, it represses lin-29A through its 5’UTR, thereby achieving isoform-specific regulation. Strikingly, we find that it is the location of the binding site that instructs the silencing mechanism, with LIN41 bound to the 5’UTR causing translational repression, while causing mRNA degradation when bound to the 3’UTR. Furthermore, we identify the transcription factor lin-29A and its co-factor mab-10 as the key targets of LIN41 for controlling self-renewal of seam cells. Because their mammalian homologs, EGR and NAB proteins, are crucial regulators of stem cell proliferation and differentiation, we hypothesize that the uncovered pathway to control seam cell self-renewal is conserved in mammalian stem cells, potentially including a LIN41-mediated regulation of EGR and NAB mRNAs. While further characterizing the four LIN41 targets, we discover that they also explain the defects of lin-41 mutants in sexual organ development. Thus, LIN41 controls the morphogenesis of the vulva through lin-29A and mab-10, and the maturation of the male tail tip through mab-3 and dmd-3. Moreover, we find that the developmental transition from a larval to an adult C. elegans epidermis depends on the upregulation of both isoforms of LIN-29. To inhibit a premature expression, lin-29A is regulated by LIN41, while the expression of lin-29B is controlled through the transcription factor HBL-1.

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