frq and qrf (Xue et al. 2014) or transcriptional
permissiveness at the frq locus (Li et al. 2015).
As the mammalian clock also contains an antisense RNA to the negative arm proteins, the
answer to this debate has implications for
clock regulation in higher eukaryotes (Li et al.
2015; Xue et al. 2014).
In addition to the transcriptional regulation
of frq, there is also regulation that occurs at the
level of translation. One mechanism arises from
the presence of non-optimal codons within
frq’s open reading frame. Among the codons
used to code for the same amino acid, some
codons occur less often than others genomewide and these are termed non-optimal codons.
Non-optimal codons correlate to low copy
numbers of a given tRNA and therefore are
thought to result in slower translation elongation rates (Zhou et al. 2013). The sequence of
frq is biased toward more non-optimal codons
and evidence suggests that this codon bias is
important for the stability of the resulting FRQ
protein. When more frequently occurring
codons were substituted into the N-terminus
of the frq open reading frame, the clock no
longer functioned (Zhou et al. 2013). As more
optimal codons are thought to lead to faster
rates of translation elongation, the loss of
rhythmicity was attributed to the improper
folding of FRQ, similar to results obtained
from the codon optimization of the negative
arm protein PER in Drosophila (Zhou et al.
2013; Fu et al. 2016). Alternatively, and to
include the more recent data showing that
FRQ is an IDP, if FRQ is translated too quickly,
it may not have a chance to bind with its stabilizing “Nanny” FRH (see also Sect. III.B.2),
which could lead to rapid degradation (Hurley
et al. 2013). Regardless of the mechanism, the
conservation of non-optimal codons within frq
appears to be essential for circadian regulation
in N. crassa and other organisms (Xu et al. 2013;
Fu et al. 2016).
A second mechanism of FRQ translational
regulation lies in the many upstream Open
Reading Frames (uORFs) of frq. The frq transcript contains an approximate 1.5 kb untranslated region at its 5
0 end (5
0 UTR) that includes
six uORFs (Garceau et al. 1997; Liu et al. 1997).
When most of the 5
0 UTR was deleted, FRQ
levels increased, suggesting that ribosome scanning through uORFs could decrease levels of
FRQ under some conditions (Liu et al. 1997).
However, unpublished experiments suggest
that the deletion of those six uORFs had no
effect on the clock’s overall rhythmicity and
did not shorten the 4 h lag between peak frq
mRNA and peak FRQ protein levels (Garceau
et al. 1997). Interestingly, uORFs are also conserved in multiple clock proteins as a regulatory mechanism in mammals (Janich et al.
2015).
2. FRQ-Interacting RNA Helicase (FRH)
Supports FRQ as a “Nanny” Protein
While all FRQ is associated with FRH, only
~40% of FRH within the cell is bound to FRQ,
with the other ~60% presumably carrying out
other functions within the cell (Cheng et al.
2005). These functions are likely related to
RNA surveillance, as FRH is a DEAD-box-type
RNA Helicase protein and is homologous to
Mtr4p in Saccharomyces cerevisiae, which
assists in RNA metabolism via its interaction
with the exosome (Lykke-Andersen et al. 2011).
FRH interacts with exosome proteins in N.
crassa (e.g., RRP44), has been found to bind
frq mRNA and influence its stability, and is
also a functional RNA helicase (Guo et al.
2009; Morales et al. 2018).
FRH has many homologs among fungal and animal
species, including some involved in the clock complex
(Cheng et al. 2005; Padmanabhan et al. 2012), and has a
well-characterized and conserved overall structure
(Conrad et al. 2016; Morales et al. 2018). Four domains
form a ring-like structure (two canonical RecA-like
domains, a small winged helix domain, and a helical
DSHCT domain) and a fifth domain forms an arch
domain or “arm” that spans across one side of the
ring and ends in a KOW or “fist” module (Conrad
et al. 2016; Morales et al. 2018). A known binding
region for FRQ is found in an additional domain (aa
100–150) within the largely unstructured N-terminus,
and the KOW/fist domain includes a binding site for
WCC interaction that can be further modulated by an
interaction with VVD (Hunt et al. 2010; Shi et al. 2010;
Hurley et al. 2013; Conrad et al. 2016; Morales et al.
2018).
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
87
permissiveness at the frq locus (Li et al. 2015).
As the mammalian clock also contains an antisense RNA to the negative arm proteins, the
answer to this debate has implications for
clock regulation in higher eukaryotes (Li et al.
2015; Xue et al. 2014).
In addition to the transcriptional regulation
of frq, there is also regulation that occurs at the
level of translation. One mechanism arises from
the presence of non-optimal codons within
frq’s open reading frame. Among the codons
used to code for the same amino acid, some
codons occur less often than others genomewide and these are termed non-optimal codons.
Non-optimal codons correlate to low copy
numbers of a given tRNA and therefore are
thought to result in slower translation elongation rates (Zhou et al. 2013). The sequence of
frq is biased toward more non-optimal codons
and evidence suggests that this codon bias is
important for the stability of the resulting FRQ
protein. When more frequently occurring
codons were substituted into the N-terminus
of the frq open reading frame, the clock no
longer functioned (Zhou et al. 2013). As more
optimal codons are thought to lead to faster
rates of translation elongation, the loss of
rhythmicity was attributed to the improper
folding of FRQ, similar to results obtained
from the codon optimization of the negative
arm protein PER in Drosophila (Zhou et al.
2013; Fu et al. 2016). Alternatively, and to
include the more recent data showing that
FRQ is an IDP, if FRQ is translated too quickly,
it may not have a chance to bind with its stabilizing “Nanny” FRH (see also Sect. III.B.2),
which could lead to rapid degradation (Hurley
et al. 2013). Regardless of the mechanism, the
conservation of non-optimal codons within frq
appears to be essential for circadian regulation
in N. crassa and other organisms (Xu et al. 2013;
Fu et al. 2016).
A second mechanism of FRQ translational
regulation lies in the many upstream Open
Reading Frames (uORFs) of frq. The frq transcript contains an approximate 1.5 kb untranslated region at its 5
0 end (5
0 UTR) that includes
six uORFs (Garceau et al. 1997; Liu et al. 1997).
When most of the 5
0 UTR was deleted, FRQ
levels increased, suggesting that ribosome scanning through uORFs could decrease levels of
FRQ under some conditions (Liu et al. 1997).
However, unpublished experiments suggest
that the deletion of those six uORFs had no
effect on the clock’s overall rhythmicity and
did not shorten the 4 h lag between peak frq
mRNA and peak FRQ protein levels (Garceau
et al. 1997). Interestingly, uORFs are also conserved in multiple clock proteins as a regulatory mechanism in mammals (Janich et al.
2015).
2. FRQ-Interacting RNA Helicase (FRH)
Supports FRQ as a “Nanny” Protein
While all FRQ is associated with FRH, only
~40% of FRH within the cell is bound to FRQ,
with the other ~60% presumably carrying out
other functions within the cell (Cheng et al.
2005). These functions are likely related to
RNA surveillance, as FRH is a DEAD-box-type
RNA Helicase protein and is homologous to
Mtr4p in Saccharomyces cerevisiae, which
assists in RNA metabolism via its interaction
with the exosome (Lykke-Andersen et al. 2011).
FRH interacts with exosome proteins in N.
crassa (e.g., RRP44), has been found to bind
frq mRNA and influence its stability, and is
also a functional RNA helicase (Guo et al.
2009; Morales et al. 2018).
FRH has many homologs among fungal and animal
species, including some involved in the clock complex
(Cheng et al. 2005; Padmanabhan et al. 2012), and has a
well-characterized and conserved overall structure
(Conrad et al. 2016; Morales et al. 2018). Four domains
form a ring-like structure (two canonical RecA-like
domains, a small winged helix domain, and a helical
DSHCT domain) and a fifth domain forms an arch
domain or “arm” that spans across one side of the
ring and ends in a KOW or “fist” module (Conrad
et al. 2016; Morales et al. 2018). A known binding
region for FRQ is found in an additional domain (aa
100–150) within the largely unstructured N-terminus,
and the KOW/fist domain includes a binding site for
WCC interaction that can be further modulated by an
interaction with VVD (Hunt et al. 2010; Shi et al. 2010;
Hurley et al. 2013; Conrad et al. 2016; Morales et al.
2018).
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
87