Most plant and animal microRNAs (miRNAs) are transcribed by RNA polymerase II. We previously discovered miRNA–like small RNAs (milRNAs) in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III) in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri–milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA–producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III–transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre–milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.
microRNAs (miRNAs) are small RNAs that are used by many organisms to regulate a wide variety of molecular, developmental, and physiological activities. In higher eukaryotes, such as animals and plants, the majority of the independent transcribed miRNAs are produced by RNA polymerase II (Pol II), an enzyme that is also responsible for the production of most of the messenger RNAs. On the other hand, only a few tRNA–associated miRNAs are known to be produced by RNA polymerase III (Pol III), an enzyme that is responsible for the production of small sized RNAs such as tRNAs and 5s rRNA. We previously identified the first fungal miRNAs by identifying the small RNAs associated with an Argonaute protein in the filamentous fungus Neurospora crassa. In this study, we examined the role of Pol II and Pol III in the production of Neurospora miRNAs. We showed that, unlike in plants and animals, Pol III appears to be a major RNA polymerase responsible for miRNA production in this fungus. Our study identified the transcriptional machinery responsible for the synthesis of fungal miRNAs and shed light on the evolutionary origin of miRNAs.