nism in which participate the three RdRP
enzymes of M. circinelloides and the Sad-3like helicase rnhA (Calo et al. 2017; Trieu et al.
2015). The Dicer function is carried out by an
atypical RNase III-like enzyme, named R3B2
(Table 5.2), which has been found only in
Mucorales. The rdRNAs have characteristics
of degradation products (Table 5.1), suggesting
that this mechanism is likely a degradation
pathway that controls the levels of specific
mRNAs (Trieu et al. 2015). This pathway regulates the expression of hundreds of highly
expressed genes involved in metabolism, regular cellular processes, and signaling; consequently, the deletion mutant lacking the key
gene r3b2 shows resistance to oxidative stress
and defects in asexual sporulation, response to
nutrient, and mating (Trieu et al. 2017). These
kinds of Dicer-independent mechanisms are
not restricted to M. circinelloides, but have
been described in other fungi. In Schizosaccharomyces pombe, Dhp exoribonuclease drives a
novel RNAi and exosome-independent pathway
of epigenetic silencing and also plays a role in
PTGS (Tucker et al. 2016). Similarly, R3B2 of M.
circinelloides plays a relevant role in the canonical silencing mechanism in parallel to its function in the Dicer-independent degradation
pathway (Trieu et al. 2015). Besides, Dicerindependent small non-coding RNAs (disiRNAs), described in N. crassa, are also produced
by a Dicer-independent RNAi pathway from
loci that generate overlapping sense and antisense transcripts as a result of convergent transcription (Lee et al. 2010). Although these
sRNAs
show
structural
characteristics
corresponding to siRNAs, none of the components of the RNAi machinery is involved in
their biogenesis (Lee et al. 2010).
The presence of esRNAs derived from
protein-coding genes has also been detected
in several fungi belonging to Ascomycota phylum, but only RNAi mutants of some species
show a phenotype that suggests the involvement of esRNAs in regulation. Different components of the RNAi machinery are involved in
light-dependent asexual reproduction and
light-independent hyphal growth in Trichoderma atroviride (Carreras-Villasen ˜or et al.
2013), in vegetative growth in Magnaporthe
oryzae (Kadotani et al. 2004), and formation
of sexual spores after sexual interaction in
Fusarium graminearum (Son et al. 2017).
Despite these remarkable examples, the number of phenotypes observed in M. circinelloides
and their strengths suggest that the RNAi pathway plays a more relevant role in early-divergent fungi than in more evolved fungi
(Ascomycota and Basidiomycota), where milRNAs and other regulatory sRNAs may fine-tune
gene expression (Villalobos-Escobedo et al.
2016).
C. Phenotypic Plasticity
In the previous sections, we described general
RNAi-mediated regulatory mechanisms that
affect the expression of hundreds of genes, but
a specific regulatory RNAi mechanism has been
revealed in M. circinelloides that target the
mRNA of a particular gene. This mechanism
was initially discovered in the screening for
spontaneous resistants to the antifungal drug
FK506 (tacrolimus). In addition to mutants in
the gene fkbA, which encodes the FK506 target
protein FKBP12, epimutant strains were
isolated that transiently silenced the expression
of fkbA (Calo et al. 2014). Interestingly, M.
circinelloides isolated from humans or other
animal hosts are able to produce a high number
of FK506-resistant epimutants (Calo et al. 2014,
2017), suggesting that this mechanism may
enable this opportunistic pathogen to readily
adapt to different environments. Silencing of
fkbA was accompanied by elevated levels of
sRNAs and required the core components of
the RNAi machinery (Calo et al. 2014, 2017).
The analysis of RNAi proteins involved in this
regulatory mechanism revealed the existence of
an epimutational RNAi pathway in M. circinelloides, which is very similar to the ex-siRNAproducing pathways (Calo et al. 2017), but the
set of RNAi proteins and their relevancy is
different, suggesting that a distinct sRNA class
is involved in this pathway (Calo et al. 2014,
2017). The generation of sRNAs depends on the
production of a dsRNA generated by the action
of one of the RdRPs (RdRP2) present in M.
circinelloides, and not from antisense transcrip5 Small RNAs in Fungi
115
enzymes of M. circinelloides and the Sad-3like helicase rnhA (Calo et al. 2017; Trieu et al.
2015). The Dicer function is carried out by an
atypical RNase III-like enzyme, named R3B2
(Table 5.2), which has been found only in
Mucorales. The rdRNAs have characteristics
of degradation products (Table 5.1), suggesting
that this mechanism is likely a degradation
pathway that controls the levels of specific
mRNAs (Trieu et al. 2015). This pathway regulates the expression of hundreds of highly
expressed genes involved in metabolism, regular cellular processes, and signaling; consequently, the deletion mutant lacking the key
gene r3b2 shows resistance to oxidative stress
and defects in asexual sporulation, response to
nutrient, and mating (Trieu et al. 2017). These
kinds of Dicer-independent mechanisms are
not restricted to M. circinelloides, but have
been described in other fungi. In Schizosaccharomyces pombe, Dhp exoribonuclease drives a
novel RNAi and exosome-independent pathway
of epigenetic silencing and also plays a role in
PTGS (Tucker et al. 2016). Similarly, R3B2 of M.
circinelloides plays a relevant role in the canonical silencing mechanism in parallel to its function in the Dicer-independent degradation
pathway (Trieu et al. 2015). Besides, Dicerindependent small non-coding RNAs (disiRNAs), described in N. crassa, are also produced
by a Dicer-independent RNAi pathway from
loci that generate overlapping sense and antisense transcripts as a result of convergent transcription (Lee et al. 2010). Although these
sRNAs
show
structural
characteristics
corresponding to siRNAs, none of the components of the RNAi machinery is involved in
their biogenesis (Lee et al. 2010).
The presence of esRNAs derived from
protein-coding genes has also been detected
in several fungi belonging to Ascomycota phylum, but only RNAi mutants of some species
show a phenotype that suggests the involvement of esRNAs in regulation. Different components of the RNAi machinery are involved in
light-dependent asexual reproduction and
light-independent hyphal growth in Trichoderma atroviride (Carreras-Villasen ˜or et al.
2013), in vegetative growth in Magnaporthe
oryzae (Kadotani et al. 2004), and formation
of sexual spores after sexual interaction in
Fusarium graminearum (Son et al. 2017).
Despite these remarkable examples, the number of phenotypes observed in M. circinelloides
and their strengths suggest that the RNAi pathway plays a more relevant role in early-divergent fungi than in more evolved fungi
(Ascomycota and Basidiomycota), where milRNAs and other regulatory sRNAs may fine-tune
gene expression (Villalobos-Escobedo et al.
2016).
C. Phenotypic Plasticity
In the previous sections, we described general
RNAi-mediated regulatory mechanisms that
affect the expression of hundreds of genes, but
a specific regulatory RNAi mechanism has been
revealed in M. circinelloides that target the
mRNA of a particular gene. This mechanism
was initially discovered in the screening for
spontaneous resistants to the antifungal drug
FK506 (tacrolimus). In addition to mutants in
the gene fkbA, which encodes the FK506 target
protein FKBP12, epimutant strains were
isolated that transiently silenced the expression
of fkbA (Calo et al. 2014). Interestingly, M.
circinelloides isolated from humans or other
animal hosts are able to produce a high number
of FK506-resistant epimutants (Calo et al. 2014,
2017), suggesting that this mechanism may
enable this opportunistic pathogen to readily
adapt to different environments. Silencing of
fkbA was accompanied by elevated levels of
sRNAs and required the core components of
the RNAi machinery (Calo et al. 2014, 2017).
The analysis of RNAi proteins involved in this
regulatory mechanism revealed the existence of
an epimutational RNAi pathway in M. circinelloides, which is very similar to the ex-siRNAproducing pathways (Calo et al. 2017), but the
set of RNAi proteins and their relevancy is
different, suggesting that a distinct sRNA class
is involved in this pathway (Calo et al. 2014,
2017). The generation of sRNAs depends on the
production of a dsRNA generated by the action
of one of the RdRPs (RdRP2) present in M.
circinelloides, and not from antisense transcrip5 Small RNAs in Fungi
115