direct evidence showed that mutants lacking
specific milRNA genes exhibited elevated levels
of the mRNAs harboring predicted target
sequences, indicating that these target
sequences are regulated by milRNAs (Lee
et al. 2010).
The discovery of milRNAs in N. crassa, a
fungus of the Ascomycota phylum, proved that
regulatory sRNAs are also produced in the
lower branches of the Eukarya domain. Subsequently, other studies revealed the presence of
similar regulatory sRNAs in fungi belonging to
both the Ascomycota (Fusarium oxysporum
(Chen et al. 2014), Penicillium chrysogenum
(Dahlmann and Ku ¨ck 2015), and Trichoderma
reesei (Kang et al. 2013)) and Basidiomycota
(Coprinopsis cinerea (Lau et al. 2013) and
Antrodia cinnamomea (Lin et al. 2015)) phyla,
suggesting that milRNAs have functional roles
in these fungal groups. However, the thoroughly accomplished studies in N. crassa and
other fungi could not identify phenotypes or
specific functions associated with mutants
enable to produce all milRNAs, such as deletion
mutants lacking dicer or ago genes, or particular milRNA. Despite the absence of phenotypes,
these studies uncovered several cases of expression profiles associated with specific developmental stages and groups of milRNAs targeting
predicted mRNAs involved in the same functional roles (Jiang et al. 2017; Lau et al. 2013,
2018; Lin et al. 2015). Other studies have also
identified milRNAs that could control the production of compounds with industrial applications, such as hydrolases and antibiotics
(Dahlmann and Ku ¨ck 2015; Kang et al. 2013).
The diversity of milRNAs across fungi,
including sequence, number, expression pattern, and especially the lack of particular phenotypes associated with mutations in these
regulators, represents a major obstacle that is
preventing the unveiling of the different roles of
milRNAs in fungal physiology. Nevertheless, all
the studies describing fungal milRNAs have
contributed to enlighten a heterogeneous
world of endogenous regulatory sRNAs in
Ascomycota and Basidiomycota phyla. The
only common feature among all these fungi is
the lack of a unique or universal pathway for
the production of milRNAs, showing a scenario
where the diversification of regulatory silencing
mechanisms is the chosen strategy rather than
the conserved pathways found in animals and
plants (Bartel 2004; Carthew and Sontheimer
2009).
B. Exon-Derived Regulatory Endogenous
Small RNAs
M. circinelloides accumulates several types of
regulatory esRNAs generated by both Dicerdependent and Dicer-independent RNAi pathways (Cervantes et al. 2013; Nicolas et al. 2010;
Trieu et al. 2015). The Dicer-dependent esRNAs, named ex-siRNAs, are derived from
exons of protein-coding genes and regulate
the expression of the producing genes by degradation of the corresponding mRNAs (Nicolas
et al. 2010). In addition to Dicer, the rest of
components of the RNAi pathway involved in
siRNA-mediated genome defense are also
implicated in the biogenesis of ex-siRNAs,
although they are combined to originate four
different classes of ex-siRNAs (Nicolas et al.
2010). The four ex-siRNA classes not only differ
in the biogenesis pathway but also in their
binding to the main Ago proteins (Ago1) and
structural characteristics, such as length, strand
bias and 5
0 nucleotide (Cervantes et al. 2013;
Nicolas et al. 2010). The ex-siRNA-mediated
regulation extends beyond the genes that produce them since hundreds of likely second targets genes are differentially expressed in
deletion mutants for genes coding for RNAi
proteins involved in ex-siRNA biogenesis
(Nicola ´s et al. 2015). The massive alteration in
gene expression in these mutants should affect
genes that participate in the developmental and
physiological processes affected in these
mutants, which include vegetative growth,
response to nutrient starvation and oxidative
stress, asexual sporulation, mating, and development (Cervantes et al. 2013; de Haro et al.
2009; Nicola ´s et al. 2007, 2015; Trieu et al. 2015).
Analysis of esRNA accumulating in Dicer
mutants in M. circinelloides revealed the existence of a new type of esRNAs, named rdRNAs
(rdrp-dependent degraded RNAs). This led to
the discovery of a non-canonical RNAi mecha114
F. E. Nicola ´s et al.
specific milRNA genes exhibited elevated levels
of the mRNAs harboring predicted target
sequences, indicating that these target
sequences are regulated by milRNAs (Lee
et al. 2010).
The discovery of milRNAs in N. crassa, a
fungus of the Ascomycota phylum, proved that
regulatory sRNAs are also produced in the
lower branches of the Eukarya domain. Subsequently, other studies revealed the presence of
similar regulatory sRNAs in fungi belonging to
both the Ascomycota (Fusarium oxysporum
(Chen et al. 2014), Penicillium chrysogenum
(Dahlmann and Ku ¨ck 2015), and Trichoderma
reesei (Kang et al. 2013)) and Basidiomycota
(Coprinopsis cinerea (Lau et al. 2013) and
Antrodia cinnamomea (Lin et al. 2015)) phyla,
suggesting that milRNAs have functional roles
in these fungal groups. However, the thoroughly accomplished studies in N. crassa and
other fungi could not identify phenotypes or
specific functions associated with mutants
enable to produce all milRNAs, such as deletion
mutants lacking dicer or ago genes, or particular milRNA. Despite the absence of phenotypes,
these studies uncovered several cases of expression profiles associated with specific developmental stages and groups of milRNAs targeting
predicted mRNAs involved in the same functional roles (Jiang et al. 2017; Lau et al. 2013,
2018; Lin et al. 2015). Other studies have also
identified milRNAs that could control the production of compounds with industrial applications, such as hydrolases and antibiotics
(Dahlmann and Ku ¨ck 2015; Kang et al. 2013).
The diversity of milRNAs across fungi,
including sequence, number, expression pattern, and especially the lack of particular phenotypes associated with mutations in these
regulators, represents a major obstacle that is
preventing the unveiling of the different roles of
milRNAs in fungal physiology. Nevertheless, all
the studies describing fungal milRNAs have
contributed to enlighten a heterogeneous
world of endogenous regulatory sRNAs in
Ascomycota and Basidiomycota phyla. The
only common feature among all these fungi is
the lack of a unique or universal pathway for
the production of milRNAs, showing a scenario
where the diversification of regulatory silencing
mechanisms is the chosen strategy rather than
the conserved pathways found in animals and
plants (Bartel 2004; Carthew and Sontheimer
2009).
B. Exon-Derived Regulatory Endogenous
Small RNAs
M. circinelloides accumulates several types of
regulatory esRNAs generated by both Dicerdependent and Dicer-independent RNAi pathways (Cervantes et al. 2013; Nicolas et al. 2010;
Trieu et al. 2015). The Dicer-dependent esRNAs, named ex-siRNAs, are derived from
exons of protein-coding genes and regulate
the expression of the producing genes by degradation of the corresponding mRNAs (Nicolas
et al. 2010). In addition to Dicer, the rest of
components of the RNAi pathway involved in
siRNA-mediated genome defense are also
implicated in the biogenesis of ex-siRNAs,
although they are combined to originate four
different classes of ex-siRNAs (Nicolas et al.
2010). The four ex-siRNA classes not only differ
in the biogenesis pathway but also in their
binding to the main Ago proteins (Ago1) and
structural characteristics, such as length, strand
bias and 5
0 nucleotide (Cervantes et al. 2013;
Nicolas et al. 2010). The ex-siRNA-mediated
regulation extends beyond the genes that produce them since hundreds of likely second targets genes are differentially expressed in
deletion mutants for genes coding for RNAi
proteins involved in ex-siRNA biogenesis
(Nicola ´s et al. 2015). The massive alteration in
gene expression in these mutants should affect
genes that participate in the developmental and
physiological processes affected in these
mutants, which include vegetative growth,
response to nutrient starvation and oxidative
stress, asexual sporulation, mating, and development (Cervantes et al. 2013; de Haro et al.
2009; Nicola ´s et al. 2007, 2015; Trieu et al. 2015).
Analysis of esRNA accumulating in Dicer
mutants in M. circinelloides revealed the existence of a new type of esRNAs, named rdRNAs
(rdrp-dependent degraded RNAs). This led to
the discovery of a non-canonical RNAi mecha114
F. E. Nicola ´s et al.