A Single dicer Gene Is Required for Efficient Gene Silencing Associated with Two Classes of Small Antisense RNAs in Mucor circinelloides

Abstract

RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2 ؊ transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.RNA-mediated gene silencing (RNA silencing) acts through transcriptional and posttranscriptional mechanisms to suppress gene expression in a sequence-specific manner. Widespread among plants, animals, and fungi, these mechanisms are involved in a wide variety of biological processes, including defense against viruses and transposons, the regulation of endogenous gene expression, and heterochromatin formation (reviewed in references 2, 5, 31, and 35). Although a number of specialized RNA-silencing pathways have been reported, a common mechanism has emerged, with homologous genes and proteins acting in different organisms. A central feature in all of these silencing phenomena is the processing of doublestranded RNA (dsRNA) (long dsRNA and pre-microRNAs [miRNAs]) into small duplexes of 21 to 25 nucleotides (nt) (small interfering RNAs [siRNAs] and miRNAs) by the RNase III enzyme Dicer. The small RNAs generated by Dicer are subsequently incorporated into the RNA-induced silencing complex (RISC), where they are used as a guide for the sequence-specific degradation of the target mRNA, the repression of its translation, or the inhibition of its transcription.The Dicer endonucleases are multidomain proteins evolutionarily conserved in eukaryotes. In metazoans and plants, in addition to two copies of the RNase III domain and a dsRNAbinding domain at the carboxyl terminus, the Dicer proteins include an amino-terminal ATP-dependent RNA helicas...

Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii

“…The upregulation expression of Mrdcl2 at the late-conidiogenesis stage may suggest that Mrdcl2 is involved in the RNAi pathway to regulate gene expression in that stage. Based on the results of sequence analysis and qRT-PCR analysis, we speculated that Mrdcl2, not Mrdcl1, is the major Dicer gene that processes dsRNA into siRNA in the RNAi mechanism in vegetative growth and development, which is consistent with what occurs in most fungi (30,32,33).…”

“…Combined with the results of bioinformatic and qRT-PCR analyses, we speculated that Mrdcl2, not Mrdcl1, is the major Dicer gene whose enzyme processes dsRNA into sRNA (miRNA or siRNA), which is consistent with what occurs in most fungi (30,32,33). Therefore, characteristics and expression profiles of sRNAs (18 to 30 nt in length) from the Δdcl2 mutant were further analyzed by high-throughput sequencing and compared to those of the WT.…”

RNA silencing in fungi

“…The efficiency of silencing using this type of plasmid was very low with only 3% of transformants showing silencing. This was dramatically improved when an intron containing hpRNA construct was used (de Haro et al, 2009). The silencing phenotype correlated with reduced levels of spliced mature mRNA of the endogenous gene, but interestingly unspliced mRNA levels remained unchanged (Nicolás et al, 2003).…”

RNA Interference in Fungi: Pathways, Functions, and Applications

“…Transformation of M. circinelloides with self-replicative plasmids or with vectors for expression of inverted repeat transgenes results in activation of gene silencing pathways and production of sRNAs of two distinct sizes, 21 nt and 25 nt (26,(67)(68)(69). Of the two Dicer proteins, DCL-2 plays the major role in siRNA generation (26). Mucor has two RdRPs, and RdRP1 is required for transgene-induced gene silencing (68).…”