Given the clear homology in structure with
other RNA helicases and the demonstrated
ATPase RNA Helicase function, it was
surprising when a strain with a point mutation
(R806H) in the KOW domain of FRH had
healthy growth but an arrhythmic clock
(Shi et al. 2010). This suggested that FRH’s
ATPase function did not explain its role in
the core clock. Further support for FRH playing dual roles within the cell came from an
additional mutant strain where FRH lost its
ATPase function yet still supported a functional clock (Hurley et al. 2013). Moreover,
the downregulation of FRH caused an ~80%
reduction in FRQ protein levels (Cheng et al.
2005). In total, this suggested that FRH plays a
structural function to support or act as a
“Nanny” protein for the intrinsically disordered protein FRQ and protect it from premature turnover (Hurley et al. 2013). Without the
stabilizing influence of FRH, FRQ phosphorylation profiles are impacted and FRQ becomes
more prevalent in the nucleus rather than the
cytoplasm, highlighting the importance of
FRH’s Nanny role for FRQ to function in the
clock (Cheng et al. 2005; Guo et al. 2010;
Cha et al. 2011; Hurley et al. 2013).
C. The Negative Arm Represses the Positive
Arm to Close the TTFL
While a great deal is known about the activation that drives the transcriptional portion of
the TTFL, the mechanistic underpinnings of
WCC repression, the “closing of the loop,” is
not as well understood. What is known is that
the FFC represses WCC transcriptional activity
at the frq locus via a direct interaction which
involves at least three key regions of FRQ
(Aronson et al. 1994; Cheng et al. 2005; He
and Liu 2005a; He et al. 2006; Guo et al. 2010).
Within the clock complex, WC-2 can interact
independently with WC-1 and FRQ and is
thought to be required for the interaction
between WC-1 and FRQ (Denault et al. 2001).
In addition, recent evidence shows that in the
dark, the “DBD” motif in WC-1 is also necessary for the interaction with the FFC (Wang
et al. 2016). Finally, FRH has also been shown
to interact independently with the WCC (Hunt
et al. 2010), highlighting the complexity of the
interactions within and between the core clock
complexes.
However, direct interactions do not
explain the functional mechanism of repression, and until recently there were two competing theories of how repression occurs. The
first potential mechanism involves the deactivation of the WCC via phosphorylation by
FRQ-associated kinases, which is thought to
lead to the repression of WCC transcriptional
activation of frq (Cheng et al. 2001b; Froehlich
et al. 2003; Schafmeier et al. 2005; He et al.
2006; Wang et al. 2019). It is known that
hyper-phosphorylated WCC has a weaker
affinity for DNA and that the WCC is not
phosphorylated unless it interacts with FRQ
(Schafmeier et al. 2005; He et al. 2006; Wang
et al. 2019). Recently, the specific clusters of
phosphorylation sites that are important for
decreased DNA binding have been identified,
with phosphorylations near the zinc-finger
binding domains of both WC-1 and WC2 being important for repression of WCC
activity (Wang et al. 2019). Additionally,
there is some support for a second, “clearance-based” model of repression. Research
has shown that oscillations in WCC phosphorylation and de-phosphorylation may also be
important for the localization of the WCC
complex (Schafmeier et al. 2008; Wang et al.
2016). There is evidence that FFC binding to
the WCC in the nucleus leads to the export of
the WCC to the cytoplasm (Hong et al. 2008).
This would suggest that repression could
occur via degradation or nuclear exclusion of
the WCC due to the physical interaction
between the WCC and the FFC (Hong et al.
2008). In the end, neither mechanism is mutually exclusive, and a two-step process has been
suggested where the WCC is removed from the
DNA (by phosphorylation) and then exported
to the cytoplasm and thereby sequestered
(Fig. 4.2) (Cha et al. 2008). Whether closure
of the clock’s negative feedback loop is
restricted to WCC phosphorylation, or also
includes nuclear export, the study of these
repression mechanisms in N. crassa has, and
will continue, to inform the understanding of
88
M. S. Jankowski et al.
other RNA helicases and the demonstrated
ATPase RNA Helicase function, it was
surprising when a strain with a point mutation
(R806H) in the KOW domain of FRH had
healthy growth but an arrhythmic clock
(Shi et al. 2010). This suggested that FRH’s
ATPase function did not explain its role in
the core clock. Further support for FRH playing dual roles within the cell came from an
additional mutant strain where FRH lost its
ATPase function yet still supported a functional clock (Hurley et al. 2013). Moreover,
the downregulation of FRH caused an ~80%
reduction in FRQ protein levels (Cheng et al.
2005). In total, this suggested that FRH plays a
structural function to support or act as a
“Nanny” protein for the intrinsically disordered protein FRQ and protect it from premature turnover (Hurley et al. 2013). Without the
stabilizing influence of FRH, FRQ phosphorylation profiles are impacted and FRQ becomes
more prevalent in the nucleus rather than the
cytoplasm, highlighting the importance of
FRH’s Nanny role for FRQ to function in the
clock (Cheng et al. 2005; Guo et al. 2010;
Cha et al. 2011; Hurley et al. 2013).
C. The Negative Arm Represses the Positive
Arm to Close the TTFL
While a great deal is known about the activation that drives the transcriptional portion of
the TTFL, the mechanistic underpinnings of
WCC repression, the “closing of the loop,” is
not as well understood. What is known is that
the FFC represses WCC transcriptional activity
at the frq locus via a direct interaction which
involves at least three key regions of FRQ
(Aronson et al. 1994; Cheng et al. 2005; He
and Liu 2005a; He et al. 2006; Guo et al. 2010).
Within the clock complex, WC-2 can interact
independently with WC-1 and FRQ and is
thought to be required for the interaction
between WC-1 and FRQ (Denault et al. 2001).
In addition, recent evidence shows that in the
dark, the “DBD” motif in WC-1 is also necessary for the interaction with the FFC (Wang
et al. 2016). Finally, FRH has also been shown
to interact independently with the WCC (Hunt
et al. 2010), highlighting the complexity of the
interactions within and between the core clock
complexes.
However, direct interactions do not
explain the functional mechanism of repression, and until recently there were two competing theories of how repression occurs. The
first potential mechanism involves the deactivation of the WCC via phosphorylation by
FRQ-associated kinases, which is thought to
lead to the repression of WCC transcriptional
activation of frq (Cheng et al. 2001b; Froehlich
et al. 2003; Schafmeier et al. 2005; He et al.
2006; Wang et al. 2019). It is known that
hyper-phosphorylated WCC has a weaker
affinity for DNA and that the WCC is not
phosphorylated unless it interacts with FRQ
(Schafmeier et al. 2005; He et al. 2006; Wang
et al. 2019). Recently, the specific clusters of
phosphorylation sites that are important for
decreased DNA binding have been identified,
with phosphorylations near the zinc-finger
binding domains of both WC-1 and WC2 being important for repression of WCC
activity (Wang et al. 2019). Additionally,
there is some support for a second, “clearance-based” model of repression. Research
has shown that oscillations in WCC phosphorylation and de-phosphorylation may also be
important for the localization of the WCC
complex (Schafmeier et al. 2008; Wang et al.
2016). There is evidence that FFC binding to
the WCC in the nucleus leads to the export of
the WCC to the cytoplasm (Hong et al. 2008).
This would suggest that repression could
occur via degradation or nuclear exclusion of
the WCC due to the physical interaction
between the WCC and the FFC (Hong et al.
2008). In the end, neither mechanism is mutually exclusive, and a two-step process has been
suggested where the WCC is removed from the
DNA (by phosphorylation) and then exported
to the cytoplasm and thereby sequestered
(Fig. 4.2) (Cha et al. 2008). Whether closure
of the clock’s negative feedback loop is
restricted to WCC phosphorylation, or also
includes nuclear export, the study of these
repression mechanisms in N. crassa has, and
will continue, to inform the understanding of
88
M. S. Jankowski et al.