Molecular determinants of miRNA target specificity and tissue-specific studies in C. elegans

MicroRNAs (miRNAs) control gene expression by repressing target messenger RNAs. Target identification is thus key to understand the biological implications of a miRNA in physiological or pathological processes, but it has remained the main challenge in the field. Traditionally, we refer to “canonical targets” when the 3’UTR of a gene contains a perfect Watson- Crick match to the 5’ sequence of the miRNA. However, many non-canonical miRNA binding sites have been identified that display seed mismatches, pairing beyond the seed or both. In this work, we aimed at understanding the molecular requirements necessary to induce silencing of a transcript by a specific miRNA in vivo. Using genome editing and physiological reporters, we focused on miRNA sharing the same seed sequence (miRNA families) as they permit to understand the involvement of both seed and non-seed pairing. For such investigation we studied the let-7 family of miRNAs because let-7 is conserved in humans and has been found implicated in several pathologies. We performed our studies in C. elegans because this miRNA family has been well characterized in this nematode, and mutant animals have obvious phenotypes that are easy to score. Our results suggest that target specificity of miRNAs belonging to a family depends on the degree of sequence complementarity between the individual miRNA and the transcript. Particularly, pairing of the 3’ sequence of the miRNA is the main determinant to establish preferential binding to a site. In addition, the seed match has a key role in modulating such specificity, as it allows to discriminate between high and low levels of miRNAs. Hence, target specificity of individual miRNAs is not hardwired, but is modulated by the miRNA abundance. We believe that our findings have a broad impact on miRNA target prediction and validation, especially if we want to invest in miRNA therapeutics. Lastly, we show that studying miRNA/target interactions in physiological settings has the power to unequivocally validate targets and expand our knowledge on the miRNA regulatory potential. In parallel, we succeeded in optimizing a FACS-based protocol to isolate worm cells, which we used to profile cell-type specific small RNAs and tissue-specific transcriptomes at single cell resolution. Given the general lack of methods to obtain primary cells and high quality tissue-specific data in the C. elegans community, such results hold the great potential to expand our knowledge about cell-type specific gene expression.