Primary miRNA (pri-miRNA) is transcribed by RNA polymerase II and has an ~85nt RNA stem-loop structure. This structure remains after the RNase III enzyme Drosha cleaves the strand approximately 22nt away from the terminal loop. The resulting pre-miRNA is about 65nt long, has a 2nt 3 overhang, and retains its stem-loop structure. It is transported out of the nucleus by Exportin-5, a nuclear export factor.

In the cytosol the pre-miRNA duplex encounters Dicer, another RNase III enzyme which binds to the 3 overhang. Dicer cleaves approximately 22nt from the base of the RNA, resulting in a miRNA duplex with no stem-loop, but a 2nt overhang at both 3 ends.

A component of the RNA-induced silencing complex (RISC), Argonaute-2, incorporates the RNA strand exhibiting weaker 5 end base-pairing into the complex. RISC then mediates base-pairing between that strand and its target mRNA. If there is very little mismatch between the pair, RISC cuts the mRNA at the point complementary to the middle of the 22nt miRNA. A high degree of mismatch between the two RNA strands causes RISC to hold onto the mRNA. In both cases the mRNA is unable to be translated and protein production is effectively inhibited.