A noncanonical Pol III-dependent, Microprocessor-independent biogenesis pathway generates a germline enriched miRNA family.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression. In canonical miRNA biogenesis, primary miRNAs are transcribed from intergenic loci or intronic regions by RNA polymerase II, sequentially cleaved by the Microprocessor complex and Dicer, and resulting mature miRNAs are loaded into Argonaute to repress target mRNAs. A minority of miRNAs are generated via noncanonical biogenesis pathways that bypass the Microprocessor and/or Dicer. Here, we describe a new Pol III-dependent, Microprocessor-independent, and Dicer-dependent biogenesis pathway exemplified by the family in . Although the family loci reside in intronic regions of protein-coding genes, we show that the miRNAs are derived from independent Pol III transcripts. Unlike other Pol III-dependent miRNAs, the family small RNAs are the dominant species derived from their loci, rather than fragments of a larger functional noncoding RNA. These germline-enriched miRNAs are loaded in multiple miRNA Argonautes, including the recently-characterized germline Argonaute ALG-5, which we demonstrate is repressive when tethered to a reporter transcript. We extend these findings, identifying additional Pol III-transcribed and noncanonical small RNAs in and human datasets, including human miR-4521. These young, noncanonical miRNAs may represent an early snapshot in the evolution of de novo miRNA genes.