Developmental small RNA transcriptomics reveals divergent evolution of the conserved microRNAmiR-100and thelet-7-complexin nematodes

Background Over the last three decades, small RNAs have emerged as one of the key post-transcriptional regulators, and are categorised into microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). Since their discovery as regulators of developmental timing in the nematode Caenorhabditis elegans , some miRNAs have been found to be highly conserved throughout animal evolution. miR-100 is one such highly conserved miRNA, possibly predating the origin of bilaterians. Furthermore, mir-100 is a member of the conserved let-7-complex , a locus containing three conserved miRNA-coding genes ( mir-100 , let-7 , and mir-125 / lin-4 ) that does not exist in the same arrangement in C. elegans . Results We performed small RNA sequencing across four developmental time-points in the satellite nematode Pristionchus pacificus , identifying 64 novel and ontogenically specifically expressed miRNAs. We also identified miR-100 as the most abundant miRNA in post-embryonic juvenile stages. Additionally, P. pacificus exhibited a novel constellation of the let-7-complex , wherein mir-100 is closely linked to let-7 , but without mir-125 / lin-4 in the same locus. Mutants of mir-100 and let-7 , individually as well as simultaneously, resulted in viable but developmentally defective worms, showing misregulation in collagen and astacin gene families. Interestingly, lin-4 mutants showed a delayed but otherwise normal development. Conclusion This study provides the first developmental small RNA transcriptome of P. pacificus , providing a community resource for future evo-devo studies. Furthermore, we identify miR-100 as the most abundant ontogenically-specific miRNA, which, along with let-7, regulates normal development in P. pacificus . Lastly, we observe two alternative arrangements of the highly conserved miRNA locus let-7-complex .