II. Protective Small RNAs
Soon after the discovery of co-suppression in
plants (Napoli et al. 1990), a similar RNAi phenomenon in response to transgenes, named
quelling, was described in Neurospora crassa.
In this mechanism, the expression of genes
harbored in the genome was suppressed when
copies of the same gene were introduced
(Romano and Macino 1992). Similar responses
to transgenes were observed in other fungi
(Nicola ´s et al. 2003; Wang et al. 2010). The
RNAi mechanism was well-characterized in N.
crassa (Chang et al. 2012), which belongs to the
Ascomycota phylum, and subsequently this
characterization was extended to other significant fungal phyla such as Mucoromycota and
Basidiomycota, represented by Mucor circinelloides (Torres-Martı ´nez and Ruiz-Va ´zquez
2017) and Cryptococcus neoformans (Feretzaki
et al. 2016; Janbon et al. 2010; Wang et al. 2012),
respectively. These analyses and others
described below revealed that RNAi pathways
have a critical role in the maintenance of the
genome integrity in response to TEs and other
invading nucleic acids. Moreover, the RNAi
mechanism as a guard of the genome soon
acquired a secondary role in the function and
evolution of centromeres, probably due to the
high abundance of TEs in these specialized
regions of the chromosomes (Friedman and
Freitag 2017; Yadav et al. 2018).
A. Small RNAs in Genome Defense
RNAi-meditated protection mechanisms that
participate in the maintenance of genome
integrity have been identified in different stages
of fungal life cycles, reflecting their importance.
1. Defense Responses During Vegetative
Growth
The RNAi mechanism that acts in the defense
against exogenous nucleic acids during vegetative growth received different names such as
quelling in N. crassa (Romano and Macino
1992), mitotic-induced silencing (MIS) in C.
neoformans (Wang et al. 2012) or gene silencing
in M. circinelloides (Nicola ´s et al. 2003). However, they constitute basically the same mechanism with particular details in each fungus that
affect the accessory RNAi proteins (Table 5.2)
and sRNAs features (Table 5.1) (Chang et al.
2012; Torres-Martı ´nez and Ruiz-Va ´zquez
2017). In these pathways, the long dsRNA is
cleaved into double-stranded small-interfering
RNAs (siRNAs) of 21–25 nucleotides, which
bind to an Ago protein in the RNA-induced
silencing complex (RISC) (Fig. 5.1). In most
studied fungal models so far, the silencing is
associated with a dramatic decrease of mature
mRNA levels from target genes, whereas those
of primary transcripts are not affected, indicating that it is mainly a post-transcriptional gene
silencing (PTGS) phenomenon (Nicola ´s et al.
2003; Pickford and Cogoni 2003).
Full understanding of the function of the
silencing mechanism in genome defense
requires the identification of the triggering signal. Quelling and MIS are initiated when multiple transgene copies are integrated in tandem
repeats in the genome (Cogoni et al. 1996;
Romano and Macino 1992; Wang et al. 2010).
This may also occur in M. circinelloides
although transgenes are maintained in selfreplicative plasmids, since in Mucorales these
plasmids are prone to form concatemers and
rearrange (Meussen et al. 2012; Michielse et al.
2004; Papp et al. 2013), which might favor the
formation of tandem repeats. How the siRNA
production is initiated in fungi is best characterized for quelling in N. crassa. Tandem
repeats alone are not sufficient to induce
siRNA production since it also requires
double-stranded breaks and homologous
recombination (HR) (Yang et al. 2015). Repetitive sequences are regions of genome instability
due to hyperrecombination events caused by
replication stress (Bzymek and Lovett 2001;
Vader et al. 2011). Thus, the proposed model
suggests that recombination intermediates produced during HR can be recognized by QDE-3,
a putative RecQ DNA helicase involved in
RNAi. This helicase might resolve the recombination intermediates into single-stranded DNA
(ssDNA), after recruiting the ssDNA-binding
complex (RPA) and the dual RdRP enzyme
5 Small RNAs in Fungi
109
Soon after the discovery of co-suppression in
plants (Napoli et al. 1990), a similar RNAi phenomenon in response to transgenes, named
quelling, was described in Neurospora crassa.
In this mechanism, the expression of genes
harbored in the genome was suppressed when
copies of the same gene were introduced
(Romano and Macino 1992). Similar responses
to transgenes were observed in other fungi
(Nicola ´s et al. 2003; Wang et al. 2010). The
RNAi mechanism was well-characterized in N.
crassa (Chang et al. 2012), which belongs to the
Ascomycota phylum, and subsequently this
characterization was extended to other significant fungal phyla such as Mucoromycota and
Basidiomycota, represented by Mucor circinelloides (Torres-Martı ´nez and Ruiz-Va ´zquez
2017) and Cryptococcus neoformans (Feretzaki
et al. 2016; Janbon et al. 2010; Wang et al. 2012),
respectively. These analyses and others
described below revealed that RNAi pathways
have a critical role in the maintenance of the
genome integrity in response to TEs and other
invading nucleic acids. Moreover, the RNAi
mechanism as a guard of the genome soon
acquired a secondary role in the function and
evolution of centromeres, probably due to the
high abundance of TEs in these specialized
regions of the chromosomes (Friedman and
Freitag 2017; Yadav et al. 2018).
A. Small RNAs in Genome Defense
RNAi-meditated protection mechanisms that
participate in the maintenance of genome
integrity have been identified in different stages
of fungal life cycles, reflecting their importance.
1. Defense Responses During Vegetative
Growth
The RNAi mechanism that acts in the defense
against exogenous nucleic acids during vegetative growth received different names such as
quelling in N. crassa (Romano and Macino
1992), mitotic-induced silencing (MIS) in C.
neoformans (Wang et al. 2012) or gene silencing
in M. circinelloides (Nicola ´s et al. 2003). However, they constitute basically the same mechanism with particular details in each fungus that
affect the accessory RNAi proteins (Table 5.2)
and sRNAs features (Table 5.1) (Chang et al.
2012; Torres-Martı ´nez and Ruiz-Va ´zquez
2017). In these pathways, the long dsRNA is
cleaved into double-stranded small-interfering
RNAs (siRNAs) of 21–25 nucleotides, which
bind to an Ago protein in the RNA-induced
silencing complex (RISC) (Fig. 5.1). In most
studied fungal models so far, the silencing is
associated with a dramatic decrease of mature
mRNA levels from target genes, whereas those
of primary transcripts are not affected, indicating that it is mainly a post-transcriptional gene
silencing (PTGS) phenomenon (Nicola ´s et al.
2003; Pickford and Cogoni 2003).
Full understanding of the function of the
silencing mechanism in genome defense
requires the identification of the triggering signal. Quelling and MIS are initiated when multiple transgene copies are integrated in tandem
repeats in the genome (Cogoni et al. 1996;
Romano and Macino 1992; Wang et al. 2010).
This may also occur in M. circinelloides
although transgenes are maintained in selfreplicative plasmids, since in Mucorales these
plasmids are prone to form concatemers and
rearrange (Meussen et al. 2012; Michielse et al.
2004; Papp et al. 2013), which might favor the
formation of tandem repeats. How the siRNA
production is initiated in fungi is best characterized for quelling in N. crassa. Tandem
repeats alone are not sufficient to induce
siRNA production since it also requires
double-stranded breaks and homologous
recombination (HR) (Yang et al. 2015). Repetitive sequences are regions of genome instability
due to hyperrecombination events caused by
replication stress (Bzymek and Lovett 2001;
Vader et al. 2011). Thus, the proposed model
suggests that recombination intermediates produced during HR can be recognized by QDE-3,
a putative RecQ DNA helicase involved in
RNAi. This helicase might resolve the recombination intermediates into single-stranded DNA
(ssDNA), after recruiting the ssDNA-binding
complex (RPA) and the dual RdRP enzyme
5 Small RNAs in Fungi
109