RNAi-deficient mutants show altered phenotypes in development or physiology. The most
remarkable exceptions to this general rule are
M. circinelloides and Trichoderma atroviride,
since mutants in core components of the
RNAi pathway show clear phenotypes associated with deregulation of large numbers of
genes (Carreras-Villasen ˜or et al. 2013; Cervantes et al. 2013; de Haro et al. 2009; Nicola ´s
et al. 2007, 2015; Trieu et al. 2015).
A. MicroRNA-Like Small RNAs
miRNAs are the most important small regulatory RNAs found in animals and plants as
they negatively regulate the expression of
hundreds of genes by either inducing degradation or repressing translation of the target
mRNAs (Carthew and Sontheimer 2009; Ghildiyal and Zamore 2009). In fungi, an in-depth
sequencing study of sRNAs interacting with
Ago proteins in N. crassa revealed a complex
system containing four different types of
sRNAs that shared several similarities with
miRNA, thereby named as miRNA-like small
RNAs (milRNA) (Lee et al. 2010). These four
classes of milRNAs were defined according to
the RNAi components that were required for
their biogenesis.
The synthesis of milRNAs type 1 (milR-1) depends on
the activity of Dicer, QDE-2, QIP, and MRPL3
(Table 5.2), whereas the synthesis of milRNAs type
2 (milR-2) only relies on the catalytic activity of QDE2, being totally independent of Dicer enzymes. \It is a
conserved protein harboring a predicted RNase III
domain similar to the well-known domains found in
Dicer enzymes. The role of MRPL3 in other organisms
is unknown, although it might be related to the fate of
mRNAs during the synthesis of proteins, considering
its association to the large subunit of the mitochondrial
ribosomes (Smits et al. 2007). Biogenesis of milRNAs
belonging to the type 3(milR-3) is quite similar to a
pathway found in plants that only depends on the
activity of Dicer enzymes (Carthew and Sontheimer
2009). The fourth type of milRNAs (milR-4) is the
most unusual class as it requires MRPL3 and is partially
dependent on Dicer enzymes (Lee et al. 2010).
Regardless of specific differences in the biogenesis of each type of milRNAs, all of them
share three main features with the synthesis of
miRNAs. First, they are produced from RNA
precursors of a single strand that forms a typical hairpin structure. Second, the dsRNA region
of the typical hairpin structure is processed to
generate two complementary sRNAs, although
only one of them is predominantly preserved
while the other is degraded. Finally, a strong
bias for uracil bases at the 5
0 termini of the
milRNA is commonly found in these sRNAs
and shared with canonical miRNAs (Lee et al.
2010). Despite these similarities, the processing
and the final features of milRNAs in N. crassa
also present several fundamental differences
compared with miRNAs in animals and plants.
Thus, milRNA genes of N. crassa are not highly
conserved in other species of fungi, indicating
an early evolution of milRNAs independent of
canonical miRNAs, which usually are highly
conserved and share sequence, function and
even tissue-specific expression in several species (Bartel 2004; Carthew and Sontheimer
2009). Additional divergences between milRNAs and miRNAs can be found at the beginning
of their biogenesis. Thus, cleavage of the
milRNA precursors (pri-milRNAs or premilRNAs) might be processed by the putative
ribonuclease MRPL3, a conserved protein harboring a predicted RNase III domain similar to
the well-known domains found in Dicer
enzymes that has not been involved in miRNA
biogenesis in plant and animals (Bartel 2004;
Carthew and Sontheimer 2009). Also, primilRNAs are transcribed by RNA polymerase
III, representing an additional significant difference compared to the well-established role of
RNA polymerase II in the transcription of primiRNAs in plants and animals (Yang et al.
2013).
The function of milRNAs has been linked
to the regulation of the expression of target
mRNAs similarly to miRNAs (Lee et al. 2010).
The first result supporting the functionality of
milRNAs was the overexpression of target
mRNAs in Dicer mutants of N. crassa, suggesting that target mRNAs are repressed by milRNAs (Lee et al. 2010). Correspondingly,
mutants devoid in the Ago protein QDE2
(Table 5.2) also displayed increased levels of
predicted target mRNAs and QDE2 coprecipitates with these target mRNAs. More
5 Small RNAs in Fungi
113
remarkable exceptions to this general rule are
M. circinelloides and Trichoderma atroviride,
since mutants in core components of the
RNAi pathway show clear phenotypes associated with deregulation of large numbers of
genes (Carreras-Villasen ˜or et al. 2013; Cervantes et al. 2013; de Haro et al. 2009; Nicola ´s
et al. 2007, 2015; Trieu et al. 2015).
A. MicroRNA-Like Small RNAs
miRNAs are the most important small regulatory RNAs found in animals and plants as
they negatively regulate the expression of
hundreds of genes by either inducing degradation or repressing translation of the target
mRNAs (Carthew and Sontheimer 2009; Ghildiyal and Zamore 2009). In fungi, an in-depth
sequencing study of sRNAs interacting with
Ago proteins in N. crassa revealed a complex
system containing four different types of
sRNAs that shared several similarities with
miRNA, thereby named as miRNA-like small
RNAs (milRNA) (Lee et al. 2010). These four
classes of milRNAs were defined according to
the RNAi components that were required for
their biogenesis.
The synthesis of milRNAs type 1 (milR-1) depends on
the activity of Dicer, QDE-2, QIP, and MRPL3
(Table 5.2), whereas the synthesis of milRNAs type
2 (milR-2) only relies on the catalytic activity of QDE2, being totally independent of Dicer enzymes. \It is a
conserved protein harboring a predicted RNase III
domain similar to the well-known domains found in
Dicer enzymes. The role of MRPL3 in other organisms
is unknown, although it might be related to the fate of
mRNAs during the synthesis of proteins, considering
its association to the large subunit of the mitochondrial
ribosomes (Smits et al. 2007). Biogenesis of milRNAs
belonging to the type 3(milR-3) is quite similar to a
pathway found in plants that only depends on the
activity of Dicer enzymes (Carthew and Sontheimer
2009). The fourth type of milRNAs (milR-4) is the
most unusual class as it requires MRPL3 and is partially
dependent on Dicer enzymes (Lee et al. 2010).
Regardless of specific differences in the biogenesis of each type of milRNAs, all of them
share three main features with the synthesis of
miRNAs. First, they are produced from RNA
precursors of a single strand that forms a typical hairpin structure. Second, the dsRNA region
of the typical hairpin structure is processed to
generate two complementary sRNAs, although
only one of them is predominantly preserved
while the other is degraded. Finally, a strong
bias for uracil bases at the 5
0 termini of the
milRNA is commonly found in these sRNAs
and shared with canonical miRNAs (Lee et al.
2010). Despite these similarities, the processing
and the final features of milRNAs in N. crassa
also present several fundamental differences
compared with miRNAs in animals and plants.
Thus, milRNA genes of N. crassa are not highly
conserved in other species of fungi, indicating
an early evolution of milRNAs independent of
canonical miRNAs, which usually are highly
conserved and share sequence, function and
even tissue-specific expression in several species (Bartel 2004; Carthew and Sontheimer
2009). Additional divergences between milRNAs and miRNAs can be found at the beginning
of their biogenesis. Thus, cleavage of the
milRNA precursors (pri-milRNAs or premilRNAs) might be processed by the putative
ribonuclease MRPL3, a conserved protein harboring a predicted RNase III domain similar to
the well-known domains found in Dicer
enzymes that has not been involved in miRNA
biogenesis in plant and animals (Bartel 2004;
Carthew and Sontheimer 2009). Also, primilRNAs are transcribed by RNA polymerase
III, representing an additional significant difference compared to the well-established role of
RNA polymerase II in the transcription of primiRNAs in plants and animals (Yang et al.
2013).
The function of milRNAs has been linked
to the regulation of the expression of target
mRNAs similarly to miRNAs (Lee et al. 2010).
The first result supporting the functionality of
milRNAs was the overexpression of target
mRNAs in Dicer mutants of N. crassa, suggesting that target mRNAs are repressed by milRNAs (Lee et al. 2010). Correspondingly,
mutants devoid in the Ago protein QDE2
(Table 5.2) also displayed increased levels of
predicted target mRNAs and QDE2 coprecipitates with these target mRNAs. More
5 Small RNAs in Fungi
113