Leptin-Responsive MiR-4443 Is a Small Regulatory RNA Independent of the Canonic MicroRNA Biogenesis Pathway

The human small RNA miR-4443 is functionally involved in several types of cancer and in the biology of the immune system, downstream of insulin and leptin signaling. Next generation sequencing evidence and structural prediction suggest that miR-4443 is not produced via the canonical Drosha–Exportin 5–Dicer pathway of microRNA biogenesis. We tested this hypothesis by using qRT-PCR to measure miR-4443 and other microRNA levels in HCT-116 cells with Drosha, Exportin 5, and Dicer knockouts, as well as in the parental cell line. Neither of the knockouts decreased miR-4443 levels, while the levels of canonical microRNAs (miR-21 and let-7f-5p) were dramatically reduced. Previously published Ago2-RIP-Seq data suggest a limited incorporation of miR-4443 into RISC, in agreement with the functional studies. The miR-4443 locus shows conservation in primates but not in other mammals, while its seed region appears in additional microRNAs. Our results suggest that miR-4443 is a Drosha, Exportin 5, and Dicer-independent, non-canonical small RNA produced by a yet unknown biogenesis pathway.