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 and sequentially cleaved by the Microprocessor complex and Dicer, and the 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 complex 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 demonstrated is repressive when tethered to a reporter transcript. We extend these findings, identifying additional Pol III transcribed and noncanonical small RNAs in and human data sets, including human miR-4521. These young, noncanonical miRNAs may represent an early snapshot in the evolution of de novo miRNA genes.