Developmental small RNA transcriptomics reveals divergent evolution of the conserved microRNA miR-100 and the let-7-complex in nematodes

Small RNAs have emerged as one of the key post-transcriptional regulators and can be broadly 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 were found to be highly conserved throughout animal evolution. miR-100 is a 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 ancient miRNA-coding genes (miR-100, let-7, miR-125/lin-4) that has been secondarily lost in C. elegans. Here, we performed small RNA-sequencing across development in the satellite nematode Pristionchus pacificus, identifying 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 lin-4 in the same locus. Knockouts of mir-100, let-7, and mir-100 let-7 double mutants in P. pacificus resulted in viable worms with developmental defects related to dysregulation of collagen. Surprisingly, lin-4 mutants showed nearly normal development. This study provides the first developmental small RNA transcriptome in P. pacificus, and indicates that the let-7-complex and small RNA pathways undergo rapid evolutionary divergence in nematodes.