Abstract
The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.
Highlights
Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans [1], our knowledge on the crucial role of endogenous small RNA as riboregulators has increased dramatically
We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs
In the basal fungus Mucor circinelloides, an opportunistic human pathogen, we previously demonstrated that biogenesis of a large group of endogenous small RNA (esRNA) molecules requires a basic RNAi machinery consisting of a Dicer-like protein, an Argonaute nuclease and two RNAdependent RNA polymerases
Summary
Since the discovery of RNAi in Caenorhabditis elegans [1], our knowledge on the crucial role of endogenous small RNA (esRNA) as riboregulators has increased dramatically. Some RNAi-competent organisms, including plants, nematodes and fungi, require RNA-dependent RNA polymerases to generate dsRNA from single-stranded RNA inducers or to amplify siRNA signals. Besides this canonical pathway, different non-canonical alternatives in which Dicer proteins do not participate have been described to be responsible for the biogenesis of specific esRNAs, the wellknown Piwi-interacting RNAs (piRNAs) and miRNAs and miRNA-like (milRNA) molecules [6,7,8]. The majority of the non-canonical miRNA molecules are poorly conserved and low in abundance, which shed doubts on their functionality
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