Published online 28 April 2010 Nucleic Acids Research, 2010, Vol. 38, No. 16 5535–5541 doi:10.1093/nar/gkq301 Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides Francisco Esteban Nicolas1, Simon Moxon 2 , Juan P. de Haro3, Silvia Calo3, Igor V. Grigoriev4, Santiago Torres-Martı´nez3, Vincent Moulton 2 , Rosa M. Ruiz-Va´ zquez3,* and Tamas Dalmay1,* 1School of Biological Sciences, 2School of Computing Sciences, University of East Anglia, Norwich, NR4 7TJ, UK, 3Departamento de Gene´ tica y Microbiologı´a, Facultad de Biologı´a, Universidad de Murcia, 30100 Murcia, Spain and 4DOE Joint Genome Institute, 2800 Mitchell Dr., Walnut Creek, CA 94598, USA Received January 10, 2010; Revised March 9, 2010; Accepted April 11, 2010 ABSTRACT Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogen- esis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eu- karyotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi. INTRODUCTION The gene silencing pathways using endogenous short RNAs (esRNAs) 20–24 nt in length are very diverse in eukaryotes (1). Most classes of esRNAs are produced from double-stranded RNA usually by a member of the Dicer family and are incorporated into an eﬀector complex containing a member of the Argonaute family (1). esRNAs that are produced from and target trans- posons seem to be the most ancient because these were found in fungi (2), plants (3) and animals (4,5). These esRNAs act in cis and may lead to DNA methylation and/or histone modiﬁcations (2,3). Another class of esRNAs, microRNAs (miRNAs), are produced from hairpin structure non-coding transcripts and target mRNAs in trans (6). miRNAs have been found in plants and animals and play a role in diverse processes including development and adaptation to environmental changes (6). Although miRNAs have not been found in fungi, dicer mutants of several fungi have been reported to be aﬀected in vegetative and developmental processes (7,8) suggesting the existence of esRNAs with regulatory func- tions in this kingdom. However, information on esRNAs in fungi is very scarce. Mucor circinelloides, a basal fungus of the clade zygomycete, has been revealed as a model organism in the fungal kingdom for the study of RNA silencing and other processes due to the availability of molecular tools and its evolutionary distance from other fungal model or- ganisms. The existence of a transgene-induced RNA silencing mechanism in M. circinelloides has been previ- ously demonstrated (9) and the pathway has been dis- sected by identifying genes and proteins involved in silencing. As a result, two dicer genes, dcl1 and dcl2, have been identiﬁed (8,10). The DCL2 protein plays a major role in gene silencing induced by sense or hairpin transgenes, being responsible for the production of two *To whom correspondence should be addressed. Tel: +34 868 887136; Fax: +34 868 883963; Email: rmruiz@um.es Correspondence may also be addressed to Tamas Dalmay. Tel: +0044 1603 593221; Fax: +0044 1603 592250; Email: t.dalmay@uea.ac.uk The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.