Target specificity and developmental functions of the let-7 microRNA

let-7 is a highly conserved microRNA(miRNA) with important functions in a wide variety of biological processes. In the nematode worm Caenorhabditis elegans (C. elegans) let-7 plays a crucial role in developmental timing, regulating temporal cell fates in the stem cell-like seam cell compartment. Study of let-7 in this context served as a useful model system for both elucidation of general principles of miRNA function and definition of universal concepts regarding developmental time and stem-cell biology. In my thesis work, I was interested in the study of molecular aspects of let-7-mediated target regulation and its developmental functions. I examined these aspects in three separate, but complementary projects. First, by performing a genome-wide RNAi screen for suppressors of let-7 lethality, I identified about 200 novel let-7 genetic interaction partners. Characterization of these genes revealed a tight connection between let-7 activity and the cell-cycle. Unfortunately, the screed did not yield any obvious and promising candidate let-7 target or upstream regulator for further study. My second project consisted of establishing a novel, quantitative in vivo miRNA target reporter system and study of let-7-mediated target regulation in different tissues of the worm. This new technique allowed the direct and quantitative visualization of miRNA activity on different targets over time, in various tissues, at a quantitative level. The main finding in these experiments was the demonstration of target specificity among different members of the let-7 family. Even in the same cell and at the same time, individual targets were differentially affected by the loss of a specific family member, showing that they have intrinsically different target specificity. I examined the sequence requirements of let-7 specificity towards lin-41 at the target site level and found that base-pairing at the 3’end of the miRNA contributes to effective and specific repression. However this is probably not sufficient, as I could not transform lin-41 repression completely to be dependent on another let-7 family member by mutating the target site. These findings have clearly further implications for our general understanding of miRNA specificity. Finally, I characterized developmental defects underlying the lethal vulva bursting phenotype of let-7 mutant worms. I showed that let-7 expression in the vulva is required for bursting suppression. Contrary to previous assumptions, let-7 is not involved in the specification of vulva precursor cells at the L3 stage, but probably has a role in later stages of vulva morphogenesis. In this context, let-60, the worm Ras homologue, is not targeted by let-7 and loss of the let-60 3’UTR and thus miRNA regulation has no functional consequences. By contrast, my experiments show robust regulation of lin-41 in all vulva cells and suggest that lin-41 is the key let-7 target in the vulva. Surprisingly, the let-7/lin-41 pathway does not influence lin-29 expression and lin-29 is, unlike in the hypodermis, not an effector of let-7/lin-41. I discovered and characterized a novel function for the heterochronic genes let-7 and lin-41 in the vulva and showed that the effectors of the heterochronic pathway can be rewired in different tissues. In sum, I used different approaches to expand our understanding of the molecular and developmental functions of the let-7 miRNA in C. elegans and my results might have further implications for the target specificity of miRNAs