A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungiby Trung Anh Trieu, Silvia Calo, Francisco E. Nicolás, Ana Vila, Simon Moxon, Tamas Dalmay, Santiago Torres-Martínez, Victoriano Garre, Rosa M. Ruiz-Vázquez

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RESEARCH ARTICLE

A Non-canonical RNA Silencing Pathway

Promotes mRNA Degradation in Basal Fungi

Trung Anh Trieu1☯, Silvia Calo1☯¤, Francisco E. Nicolás1, Ana Vila1, Simon Moxon2,

Tamas Dalmay3, Santiago Torres-Martínez1, Victoriano Garre1, Rosa M. Ruiz-Vázquez1* 1 Department of Genetics and Microbiology, University of Murcia, Murcia, Spain, 2 The Genome Analysis

Centre, University of East Anglia, Norwich, United Kingdom, 3 School of Biological Sciences, University of

East Anglia, Norwich, United Kingdom ☯ These authors contributed equally to this work. ¤ Current address: Department of Basic Sciences, PUCMM, Santiago, Dominican Republic * rmruiz@um.es

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 fungusMucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share commonmachinery 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 sRNAmolecules 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 theMucor 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 betweenmRNA degradation and post-transcriptional gene silencing.

Author Summary

Most eukaryotic organisms produce different classes of endogenous small RNA (esRNA) molecules that suppress gene expression through RNA interference (RNAi) pathways. These

PLOSGenetics | DOI:10.1371/journal.pgen.1005168 April 13, 2015 1 / 32

OPEN ACCESS

Citation: Trieu TA, Calo S, Nicolás FE, Vila A, Moxon

S, Dalmay T, et al. (2015) A Non-canonical RNA

Silencing Pathway Promotes mRNA Degradation in

Basal Fungi. PLoS Genet 11(4): e1005168. doi:10.1371/journal.pgen.1005168

Editor: Geraldine Butler, University College Dublin,

IRELAND

Received: January 20, 2015

Accepted: March 20, 2015

Published: April 13, 2015

Copyright: © 2015 Trieu et al. This is an open access article distributed under the terms of the

Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All raw reads files of

Mucor circinelloides small RNAs are available from the GEO database (accession numbers GSM469403,

GSM469406 and GSM469407) and from the SRA database (accession numbers SRR835448 and

SRR1576768).

Funding: This work was supported by the Spanish

Ministerio de Ciencia e Innovación (BFU2009-07220, co-financed by FEDER, to RMRV); and the Spanish

Ministerio de Economía y Competitividad (BFU201232246, co-financed by FEDER, to VG). TAT was funded by the MOVER Program of the European

Union (Erasmus Mundus Action 2). FEN was funded pathways, which may differ among organisms, are normally involved in genome defense, heterochromatin formation and regulation of genes involved in multiple cellular functions.

In the basal fungusMucor circinelloides, an opportunistic human pathogen, we previously demonstrated that biogenesis of a large group of esRNAmolecules requires a basic RNAi machinery consisting of a Dicer-like protein, an Argonaute nuclease and two RNAdependent RNA polymerases. This canonical dicer-dependent pathway regulates different cellular processes, such as vegetative sporulation. Besides those esRNAs generated by this canonical RNAi pathway, we have identified a new rdrp-dependent dicer-independent esRNA class. These esRNAs are produced by a degradation pathway in which the RdRP proteins signal specific transcripts that will be degraded by a newly identified RNase. This RNase, named R3B2, presents unique domain architecture, can only be found in basal fungi and it is also involved in the canonical dicer-dependent RNAi pathway. Our results expand the role of RdRPs in gene silencing and reveal the involvement of these proteins in a new RNA degradation process that could represent the first step in the evolution of RNAi.

Introduction

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. Multiple classes of esRNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been identified both in metazoans and lower eukaryotic organisms [2–5]. Biogenesis of most of those esRNAs shares a minimal common machinery consisting in an RNase III endonuclease

Dicer that processes double-stranded RNA (dsRNA) precursors into small RNA (sRNA) molecules, and an Argonaute endonuclease that binds sRNAs and uses them as a guide to identify and cleave complementary target mRNA. Additionally, 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, not only the wellknown Piwi-interacting RNAs (piRNAs) but also miRNAs and miRNA-like (milRNA) molecules [6–8]. In these cases, the catalytic activity of Argonaute family proteins and the trimming activity of specific exonucleases are required for the production of mature esRNAs. However, the majority of the non-canonical miRNA molecules are poorly conserved and low in abundance, which shed doubts on their functionality.