A Mammalian Herpesvirus Uses Noncanonical Expression and Processing Mechanisms to Generate Viral MicroRNAs

Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA polymerase II and then processed by the Drosha endonuclease to generate ∼60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer to generate mature miRNAs. Previously, some short introns, called miRtrons, were reported to fold into pre-miRNA hairpins after splicing and debranching, and miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin RNAs. Here we report that the miRNAs encoded by murine γ-herpesvirus 68 (MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs are transcribed from RNA polymerase III promoters located within adjacent viral tRNA-like sequences. The resultant pri-miRNAs, which bear a 5′ tRNA moiety, are not processed by Drosha but instead by cellular tRNase Z, which cleaves 3′ to the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to yield the mature viral miRNAs. ► Canonical microRNAs are transcribed by Pol II and processed by Drosha and Dicer ► MHV68 microRNAs are instead transcribed by Pol III and processed by tRNaseZ and Dicer ► MHV68-encoded primary microRNAs consist of tRNAs linked to pre-microRNA hairpins ► tRNaseZ cleavage removes the tRNA and frees the pre-microRNA for Dicer processing