311
Commentary
Post-transcriptional control of circadian rhythms
Shihoko Kojima, Danielle L. Shingle and Carla B. Green*
Department of Neuroscience, University of Texas Southwestern Medical Center, NB4.204G, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
*Author for correspondence ([email protected])
Journal of Cell Science 124, 311-320
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.065771
Summary
Circadian rhythms exist in most living organisms. The general molecular mechanisms that are used to generate 24-hour rhythms are
conserved among organisms, although the details vary. These core clocks consist of multiple regulatory feedback loops, and must be
coordinated and orchestrated appropriately for the fine-tuning of the 24-hour period. Many levels of regulation are important for the
proper functioning of the circadian clock, including transcriptional, post-transcriptional and post-translational mechanisms. In recent
years, new information about post-transcriptional regulation in the circadian system has been discovered. Such regulation has been
shown to alter the phase and amplitude of rhythmic mRNA and protein expression in many organisms. Therefore, this Commentary
will provide an overview of current knowledge of post-transcriptional regulation of the clock genes and clock-controlled genes in
dinoflagellates, plants, fungi and animals. This article will also highlight how circadian gene expression is modulated by posttranscriptional mechanisms and how this is crucial for robust circadian rhythmicity.
Journal of Cell Science
Key words: Circadian, mRNA decay, Translation, Rhythmic, mRNA stability, Splicing
Introduction
The rotation of the earth creates a daily fluctuation of environmental
cues. Organisms have evolved internal timing systems, called
circadian clocks (see Box 1 for a glossary of terms), to coordinate
their daily activities to anticipate and prepare for these
environmental changes. Circadian rhythms are endogenous and
persist under constant conditions, such as constant darkness, with
a period (Box 1) of ~24 hours. The clock can also be reset by
external cues, such as light and temperature, to maintain synchrony
with the environment. Circadian clocks govern many biological
processes, ranging from molecular and biochemical pathways to
physiological and behavioral rhythms. Conceptually, there are three
main components to a functional circadian clock (Fig. 1A). In
order for an organism to properly synchronize its internal clock to
its external environment, it must perceive a signal from the
environment (input) and adjust the timing of the clock accordingly.
These signals feed into the molecular clock (oscillator), which
consists of several clock genes that form transcriptionaltranslational feedback loops. This oscillator generates self-sustained
rhythms of ~24 hours. Finally, the timing information is transmitted
to different pathways downstream of the molecular clock, which
control a variety of different molecular and physiological processes
that exhibit circadian rhythmicity (output).
Genetic and molecular approaches have identified cellular
regulatory mechanisms that are necessary for accurate clock
function in many organisms. One common property of an oscillation
is to escape from equilibrium; forming a negative feedback loop is
one way to achieve this temporary departure from equilibrium.
Although the details vary among organisms, circadian clocks
generally utilize interlocking transcriptional-translational feedback
loops as a mechanism to generate rhythms (Fig. 1B). The similarity
among all of these systems strongly suggests that this feedback
loop reflects a common lineage of circadian oscillators (Wijnen
and Young, 2006). The molecular clock drives the rhythmic
expression of core clock genes, together with a broad array of
other circadian-controlled genes (ccgs) that are involved in various
output pathways. Transcriptome analyses revealed that the
expression of ~5–10% of genes exhibits circadian rhythmicity at
steady-state mRNA levels in any given organism or tissue (Duffield,
2003). For transcripts to be rhythmic, they are required to have
relatively short half-lives. A theoretical model of how mRNA
stability impacts their amplitude (Box 1) shows that the more
stable the transcript, the lower the amplitude of its cycling (Wuarin
et al., 1992). Therefore, the regulation of mRNA stability, or its
decay rate, plays an important role in the cycling of transcripts. In
addition to mRNA decay, other post-transcriptional mechanisms
also play an important role in rhythmic expression. For example,
in some cases, protein synthesis rhythms can be uncoupled from
mRNA rhythms, suggesting that translation can also be a key
regulatory node. Proteomic analysis in mouse liver revealed that
up to 20% of soluble proteins show rhythmic expression, although
almost half of these cycling proteins did not exhibit corresponding
rhythmic mRNA expression (Reddy et al., 2006). In this
Commentary, we will review the evidence for post-transcriptional
regulation in dinoflagellates, plants, fungi and animal circadian
clocks to elucidate why and how circadian clocks have incorporated
post-transcriptional regulation into the clock-generating machinery.
Post-transcriptional regulation
The control of gene expression is a complex process. Even after
mRNA is transcribed from DNA, mRNAs can undergo many
processing and regulatory steps that influence their expression
(Keene, 2010). This type of regulation, at the post-transcriptional
level, is beneficial, because it allows cells to alter protein levels
rapidly without requiring transcript synthesis or processing. mRNA
processing begins immediately following transcription with splicing
and, in some cases, results in multiple isoforms of the transcript.
Upon translocation into the cytoplasm, some mRNAs directly
undergo deadenylation and degradation, whereas others can be
stored in cytoplasmic compartments, such as processing bodies or
stress granules, until they are degraded or re-polyadenylated. Other
transcripts interact with the translational machinery and are
translated into protein immediately after localization to the
cytoplasm. Other mRNAs interact with localization machinery to
312
Journal of Cell Science 124 (3)
A
B
Negative regulators
Positive regulators
Input
Oscillator
Output
ccg
C
Output
Exosome
Transcription
Exon 1
Exon 2
Alternative splicing
Degradation
Exon 3
Storage and
silencing
Splicing
m7Gppp
Journal of Cell Science
m7Gppp
AAAAA
A
m7Gppp
miRNA
AAAAA
STOP
AAAAAAA
Export
m7
Gppp
RBP
Localization
and transport
Translation
RBP
RBP
AAAAAAA
Poly(A) tail length
A
Deadenylation
Ribosome
m7Gppp
AAA
m7Gppp
AAAAAA
Polyadenylation
m7Gppp
A
AA
A
AAAAA
Fig. 1. Circadian clocks and their contribution to post-transcriptional events. (A)The circadian system is generally considered to comprise three major
components: input, oscillator and output. Light is the most powerful input signal, but other cues, such as temperature and nutrient availability, are also used.
Examples of output include hormone secretion, body temperature, metabolic activity, sleep and wake cycle (mammals), eclosion, locomotor activity, olfactory
responses (Drosophila), conidiation, carbon and nitrogen metabolism (Neurospora), flowering, germination, leaf movement and photosynthesis (Arabidopsis).
(B)The generic structure of the molecular clock. Heterodimeric positive regulators (blue and green) activate the expression of negative regulators (purple and pink)
by binding cis-regulatory elements in their promoters. Upon translation, these negative regulators inhibit their own transcription by blocking the activity of the
positive regulators to complete the cycle. This cycle takes about 24 hours and thus generates circadian oscillation. Positive regulators also activate transcription of
ccgs to drive rhythmic gene expression and generate circadian output. (C)The involvement of circadian rhythms in post-transcriptional regulation. Red oscillators
indicate the steps that are known to be regulated by circadian clocks. Green, yellow and red arrows indicate pathways leading towards translation, translational
silencing and mRNA degradation pathways, respectively. m7Gppp, 7-methylguanosine cap.
be transported to a specific compartment within the cell for
localized translation (Fig. 1C). All of these steps must be
coordinated to ensure the appropriate expression profiles of mRNAs
and proteins. In general, these events are mediated by cis-elements
commonly found in 5⬘ or 3⬘ untranslated regions (UTRs) of
mRNAs, which serve as specific binding sites for trans-acting
factors, such as RNA-binding proteins (RBPs) or microRNAs
(miRNAs). The fate of the mRNA is therefore largely determined
by the composition of these trans-acting factors and the timing of
the interaction.
Post-transcriptional regulation in
dinoflagellates
The very first evidence regarding post-transcriptional regulation of
the circadian clock came from the study of Lingulodinium (formerly
known as Gonyaulax). In this primitive eukaryotic unicellular
organism, several different physiological processes are regulated
by circadian rhythms, such as photosynthesis, cell motility, cell
division and luminescence flashing (Hastings, 2007). Lingulodinium
has a highly developed capacity for bioluminescence and is one of
the main species contributing to nocturnal luminescence of the sea.
This nocturnal bioluminescence is produced from specialized
bioluminescent organelles, the scintillons, which contain three
components that are necessary for the chemical reaction: the
substrate luciferin, a luciferin-binding protein (LBP) and the
enzyme luciferase (LCF) (Morse et al., 1989). The clock controls
the synthesis of these proteins, and both LBP and LCF are
rhythmically expressed and peak at night, causing night-time
flashing. However, the expression of lbp mRNA is not rhythmic,
demonstrating that LBP and LCF expression is posttranscriptionally controlled (Johnson et al., 1984; Morse et al.,
1989). Further research showed that an RNA-binding protein,
clock controlled translational regulator (CCTR), interacts with a
UG-repeat sequence in the lbp 3⬘-UTR and represses lbp translation
Post-transcriptional control of circadian rhythms
Journal of Cell Science
during the day (Mittag et al., 1994). A similar protein, CHLAMY1,
has been identified in Chlamydomonas. It also interacts with an
UG repeat in a circadian-dependent manner (Mittag, 1996),
indicating that this phenomenon is conserved among at least some
algae.
Post-transcriptional regulation in plants
One of the first circadian phenomena recorded was in plants, in the
18th century, when leaf movement was found to vary between
night and day, even in constant conditions. Since then, many more
circadian phenomena have been described and Arabidopsis has
emerged as an important model system for the discovery of plant
clock mechanisms. The Arabidopsis clock consists of at least three
interlocking feedback loops, the so-called ‘morning’, ‘evening’
and ‘core’ loops (Pruneda-Paz and Kay, 2010) (Fig. 2). The core
loop is driven by two negative regulators, CIRCADIAN CLOCK
ASSOCIATED 1 (CCA1) and LATE ELONGATED
HYPOCOTYL (LHY), and one positive regulator, TIMING OF
CAB 1 (TOC1). There are several photoreceptors in plants
and, among them, CRYPTOCHROME 1 and 2 as well as
PHYTOCHROME B are regulated by the circadian clock to provide
light information to the central oscillator (Chen et al., 2004). Light
induces the expression of CCA1 and LHY (as well as a number of
ccgs), and this induction can entrain the clock (Martinez-Garcia et
al., 2000; Wang and Tobin, 1998). Light also regulates posttranscriptional events. For example, the stability of CCA1 mRNA
is different between the dark and light phases, indicating that light
(or lack of light) controls the transcript stability (Yakir et al.,
2007). Furthermore, light upregulates the level of LHY protein
under conditions in which it does not alter the level of LHY
transcript (Kim et al., 2003). Lastly, blocking the ZEITLUPE
(ZTL)-mediated light input pathway alters the amplitude of
rhythmic TOC1 protein without changing its mRNA amplitude.
ZTL is another blue-light photoreceptor and forms a protein
complex with GIGANTEA (GI) to regulate TOC1 protein
expression (Kim et al., 2007b). These results indicate that light not
only plays a pivotal role in activating transcription, but also
regulates the stability of the transcript and its rhythmic expression
pattern.
Recent microarray studies revealed that clock-controlled genes
in Arabidopsis have short half-lives and their rapid mRNA decay
is mediated by a downstream (dst) element in the 3⬘-UTR (Gutierrez
et al., 2002; Perez-Amador et al., 2001). Moreover, a mutant strain
(dst1) was recently isolated, in which the dst-mediated decay
pathway is disrupted. This mutant shows abnormal daily cotyledon
movement (Lidder et al., 2005), further indicating the importance
of the regulation of transcript stability to the circadian clock. The
circadian clock also controls splicing in Arabidopsis (see section
below on temperature-induced alternative splicing).
Post-transcriptional regulation in Neurospora
Neurospora crassa has been an important fungal model system for
understanding the molecular control of circadian rhythms,
combining powerful genetics with easily measured circadian
outputs such as the daily conidiation rhythm – the asexual
reproduction of spores. The molecular clock in Neurospora is
composed of two positive regulators, White Collar-1 (WC-1) and
White Collar-2 (WC-2), which together form the WC protein
complex (WCC) and activate the transcription of frq, a core
component of the clock that encodes a negative regulator, as well
as a number of ccgs. Upon translation, FRQ forms a complex with
PHYB
C s
CRYs
PHYB CRYss
PHYB
313
CRYs
C
PRR7 PRR9
Morning
m7Gppp
m7Gppp
m7Gppp
AAAA
AAAA
AAAA
CCA1
CCA1 LHY
LHY
ZTL
GI
Core
TOC1
TOC1
Evening
GI
Fig. 2. The effect of light on post-transcriptional regulation of clock genes
in Arabidopsis. Light is perceived by photoreceptors (PHYB, red light; CRY1
and CRY2; blue light) and this input signal is transmitted to the circadian core
loop to entrain the clock. ZTL is also a blue-light photoreceptor, affecting an
evening loop, and regulates TOC1 expression together with GIGANTEA (GI).
Light also provokes several post-transcriptional events, such as CCA1 mRNA
degradation, translational activation of LHY and the enhancement of the
amplitude of TOC1 rhythmic expression. PRR7 and PRR9 are PSEUDO
RESPONSE REGULATOR 7 and PSEUDO RESPONSE REGULATOR 9.
FRQ-interacting RNA helicase (FRH) and inhibits WCC activity
by promoting the phosphorylation of WCC. This leads to inhibition
of its own mRNA synthesis, thereby closing the feedback loop
(Brunner and Kaldi, 2008). Among these four core clock genes,
only frq exhibits rhythmic expression at both mRNA and protein
levels (Brunner and Kaldi, 2008), and the rhythmic expression of
FRQ contributes to the rhythmic expression of WC-1 protein (Lee
et al., 2000). The peak of cycling FRQ levels is 4–6 hours after its
mRNA peak (Garceau et al., 1997). This led to the hypothesis that
FRQ itself might also play a role in the degradation of its own
mRNA, particularly when the mRNA level is declining and the
protein level is increasing. In fact, the FRQ–FRH protein complex
(FFC) destabilizes the frq mRNA by shortening the length of its
poly(A) tail and also recruits the exosome. It is not known whether
FFC formation or its mRNA-destabilizing activity is phase
dependent (Guo et al., 2009). The exosome has several functions,
including RNA degradation (Belostotsky, 2009), and it is often
referred to as an analog of the proteasome, which is part of the
protein degradation machinery. The idea that the exosome is
essential for appropriate regulation of the Neurospora circadian
clock is supported by the following data. Firstly, the rhythmic
expression of rrp44, one of the core catalytic subunits of the
exosome, is controlled by WCC. Secondly, knocking down rrp44
abolishes the conidation rhythm and lengthens the period of
rhythmic expression of frq and FRQ. Finally, RRP44 interacts with
FFC (Guo et al., 2009). None of the identified core clock proteins
in other organisms are known to directly interact with the exosome,
but this has not been tested in other systems and it would be
interesting to see whether this mechanism is conserved.
Another unique phenomenon in Neurospora is the existence of
a long antisense RNA of frq (about 5 and 5.5 kb) (Kramer et al.,
2003). This antisense frq RNA shows rhythmic expression and its
expression pattern is anti-phase to that of the sense frq RNA. The
function of this antisense RNA is not clear, but mutant strains
lacking the expression of this antisense RNA have a delayed frq
314
Journal of Cell Science 124 (3)
Journal of Cell Science
mRNA rhythm and an altered response to light pulses (Kramer et
al., 2003). It is also not known whether the antisense frq, or a
possible RNA duplex of sense and antisense frq, is subject to FFCmediated RNA degradation by the exosome.
Post-transcriptional regulation in animals
Genetic analysis of rhythms in Drosophila began in the early
1970s. Period (Per) was the first gene to be identified and then
cloned a decade later (Bargiello and Young, 1984; Konopka and
Benzer, 1971; Reddy et al., 1984). It took another decade for the
mammalian homologue of Per to be identified and cloned (Sun et
al., 1997; Tei et al., 1997). The Drosophila and mouse clock
mechanisms are, generally speaking, conserved, although some of
the genes are different and the mammalian system is more complex,
containing multiple versions of some of the core clock genes.
Detailed descriptions of the molecular clock structure in flies and
mammals can be found elsewhere (Hastings et al., 2008; Wijnen
and Young, 2006), but in brief, a heterodimeric transcription factor
complex – called CLOCK and CYCLE (CYC) in flies and CLOCK
and BMAL1 in mammals – activates the expression of genes that
encode components of a repressive complex – PER and TIMELESS
(TIM) in flies, and PER and CRYPTOCHROME (CRY) in
mammals. The PER–TIM or PER–CRY complexes repress the
activity of CLK–CYC (or of CLOCK–BMAL1), which closes the
loop. In mammals, REV-ERB, a member of the REV-ERB family
of nuclear receptors, binds to the promoter region of Bmal1 and
also regulates CLOCK–BMAL1 activity by inhibiting Bmal1
transcription. This forms a second feedback loop (Drosophila also
has a similar interlocking loop, composed of different genes, but
will not be discussed here).
The first examination of the post-transcriptional regulation of
clock genes in animals was a detailed comparison of the active
transcription and the steady-state expression profiles of Per. This
analysis revealed that the half-life of Per mRNA changes
throughout the circadian cycle, indicating that the stability of Per
mRNA is under circadian control (So and Rosbash, 1997). It was
subsequently shown that cycling mRNAs in mammals, such as
Per2 and Cry1, also change their mRNA stability around the
clock, and are more stable during the rising phase of mRNA
cycling and less stable during the declining phase (So and
Rosbash, 1997; Woo et al., 2010; Woo et al., 2009). As a similar
regulation has been described for CCA1 in Arabidopsis (Yakir et
al., 2007), this mechanism is conserved at least in animals and
plants. The first evidence in mammals for post-transcriptional
regulation came from a study showing that Per1 mRNA decays
more rapidly following light induction than that of the control
luciferase gene, which was driven by the Per1 promoter in the
central clock in the brain (Wilsbacher et al., 2002). A later study
demonstrated that an element in the 3⬘-UTR of Per1 promotes its
instability, thereby contributing to this destabilization (Kojima et
al., 2003). The 3⬘-UTRs of other clock genes, such as Drosophila
Per, rat Aanat, and mouse Per2, Per3 and Cry1, also contribute
to regulating their rhythmic expression, thereby leading to an
accurate circadian period (Chen et al., 1998; Kim et al., 2005;
Kim et al., 2007a; Kwak et al., 2006; Woo et al., 2010; Woo et
al., 2009).
Several trans-factors (mostly RBPs) are known to regulate the
clock or clock-controlled genes. LARK (or RBM4) is an RNAbinding protein that contains three RNA-binding domains (Kojima
et al., 2007; Lai et al., 2003; Newby and Jackson, 1993). In
Drosophila, Lark affects locomotor activity and eclosion rhythms
(Huang et al., 2009; Newby and Jackson, 1996; Schroeder et al.,
2003), and activates the translation of Eip74EF, an ecdysoneinduced protein. The molecular function of Eip74EF has not been
clarified, but an Eip74EF mutant fly exhibits an early eclosion
phenotype (Huang et al., 2007). This is reminiscent of lark mutant
flies, which also have an abnormally early eclosion (Newby and
Jackson, 1993), suggesting that the post-transcriptional control of
Eip74EF by LARK is essential for eclosion rhythms. In mammals,
LARK1 (or RBM4a) activates the translation of PER1 by
interacting with the Per1 3⬘-UTR without affecting its mRNA
level (Kojima et al., 2007). The phase of LARK expression is
similar to that of PER1 expression in the mouse suprachiasmatic
nuclei (SCN), a circadian pacemaker (Box 1) in mammals, and
peaks about 4–6 hours after its mRNA peak. The parallel timing of
PER1 and LARK expression suggests that their interaction is
important, although the function of mouse LARK in vivo is yet to
be determined. There has also been a report that LARK activates
internal ribosome entry site (IRES)-mediated translation, whereas
it suppresses cap-dependent translation (Lin et al., 2007), possibly
through the interaction with Argonaute proteins (Hock et al., 2007;
Lin et al., 2007). Together, this further indicates the role of LARK
in translational control. Interestingly, the expression of LARK
itself is also under post-transcriptional control. LARK protein
levels are rhythmic, whereas its mRNA levels remain unchanged
around the clock in both mammals and flies (Kojima et al., 2007;
McNeil et al., 1998).
Another group of RBPs that play significant roles in the circadian
system is the heterogeneous nuclear ribonucleoproteins (hnRNPs).
More than 30 proteins have been categorized as hnRNPs; each
individual hnRNP has more than one function in the posttranscriptional events described in Fig. 1C (Chaudhury et al.,
2010). Among these, three hnRNPs seem to be particularly relevant
to the circadian clock system: hnRNP I, hnRNP D and hnRNP Q.
hnRNP I, also called polypyrimidine-tract-binding protein (PTB),
has four RNA-recognition motifs (RRMs) and binds to sequences
that contain 15 to 25 pyrimidines, with a preference for pyrimidine
tracts that have a UCUU sequence element (Singh et al., 1995).
Interestingly, hnRNP I shuttles between the cytoplasm and nucleus
(Sawicka et al., 2008), and the protein level of hnRNP I in the
cytoplasm is rhythmic, even though its overall level is not (Kim et
al., 2010). hnRNP I interacts with the 3⬘-UTR of Per2 mRNA and
promotes its degradation (Woo et al., 2009) (Fig. 3A). hnRNP I
also interacts with an IRES of Rev-erb mRNA and activates its
translation (Kim et al., 2010). The fluctuation of cytoplasmic
hnRNP I is almost anti-phase to Per2 mRNA expression (Fig. 3A)
and to REV-ERB mRNA and protein expression (Kim et al.,
2010; Woo et al., 2009). Therefore, the activity of hnRNP I might
only have a minor effect on REV-ERB translation, but this is a
good example of hnRNP proteins having multiple functions. A
similar observation has been reported for the interaction between
hnRNP D and Cry1 (Woo et al., 2010) (Fig. 3A). hnRNP D, also
known as ARE-binding factor 1 (AUF1), recognizes a U-rich
sequence of the Cry1 3⬘-UTR and destabilizes its mRNA. The
rhythmic expression of cytoplasmic hnRNP D is anti-phase to
Cry1 mRNA expression (Woo et al., 2010), supporting the idea
that Cry1 mRNA stability is negatively controlled by hnRNP D
(Fig. 3A). Although these data are intriguing, the roles of hnRNP
proteins are complex and a complete understanding of their
functions in the clock system will require additional mechanistic
insights. For example, AUF1 has been implicated in both
stabilization and destabilization of target mRNAs, depending on
Post-transcriptional control of circadian rhythms
A
B
AANAT
Cry1 and Per2
hnQ
m7
AAAAA
Gppp
hnQ
Q
IRES
AAAAA
m7Gppp
Ribosome
m7
Gppp
m7
m7 Gppp
Gppp
m7
Gppp
AAAAA
AAAAA
AAAAA
AAAAA
hnQ hnQ
hnQ
hnQ
m7Gppp
m7
AAAAA
Gppp
AANAT
AANAT
T
hnQ
Stabilization
hnRNPD/I
P
AANAT
A
ANAT
T
P
AANAT
AANAT
T
hnQ
hnQ
hnQ hnQ
AAAAA
Degradation
P
AANAT
AANAT
T
P
AANAT
AANAT
T
P
AANAT
AANAT
T
P
AANAT
A
ANAT
T
hnQ
AANAT
AANAT
T
PKA
P
AANAT
AANAT
T
Degradation
Journal of Cell Science
315
Tryptophan
Melatonin
Fig. 3. Post-transcriptional control of mammalian clock genes. (A)The regulation of Cry1 or Per2 mRNA stability by hnRNP D or I (hnD/I). During the nighttime, the expression of hnRNP proteins in the cytoplasm is relatively high and more Cry1 and Per2 mRNAs are subject to degradation, leading to lower levels of
mRNA (relative RNA levels depicted by curves on the left). By contrast, the level of hnRNPs is relatively low during the day; therefore, both Cry1 and Per2
mRNA can escape from the degradation caused by hnRNPs. Thus, these mRNAs become more stable, leading to higher mRNA levels. (B)Post-transcriptional and
post-translational regulation of AANAT protein expression. During the night-time, hnRNP Q (hnQ) expression increases and this promotes the translation of
AANAT through the interaction with IRESs. AANAT protein is phosphorylated by a PKA-mediated pathway and this prevents it from being degraded by the
proteasome. Both of these post-transcriptional and post-translational regulatory steps contribute to the extreme amplitude of AANAT expression during the night.
At the same time, hnRNP Q also binds to the 3⬘-UTR of Aanat mRNA and promotes degradation of Aanat mRNA. During the day, Aanat mRNA expression and
the level of cytoplasmic hnRNP Q are low, and little or no AANAT protein is made. Melatonin is synthesized from tryptophan by four enzymatic steps, of which
AANAT catalyzes the third to convert serotonin to N-acetylserotonin. The effects of hnRNP L and hnRNP R are not depicted here for simplicity. m7Gppp, 7methylguanosine cap.
the circumstances, and, moreover, it exists in four alternatively
spliced isoforms, which have different activities (Raineri et al.,
2004). It will be interesting to learn whether one or more of these
isoforms is circadian and, if so, whether this varies between tissues,
as well as which of these isoforms contribute to the control of
clock gene expression.
hnRNPs also function in the melatonin synthesis pathway, a key
rhythmic output of the circadian clock. Melatonin is produced by
the pineal gland and its levels exhibit circadian rhythmicity in
amphibians, birds, reptiles and mammals, with a peak during the
dark phase (Iuvone et al., 2005). The nocturnal increase in
circulating melatonin is mirrored by the expression of
arylalkylamine N-acetyltransferase (AANAT). AANAT is the ratelimiting enzyme in the melatonin biosynthetic pathway and its
expression is rhythmic, with a more than 100-fold increase in
amplitude during the night (Roseboom et al., 1996). However, the
expression pattern of Aanat mRNA is species specific; it is rhythmic
in rodents and peaks a few hours prior to the AANAT protein, but
remains constant around the clock in primates and ungulates.
Regardless of this difference, one common principle is that mRNA
expression is out of phase compared with its protein levels,
indicating that Aanat is under post-transcriptional control.
Phosphorylation of AANAT mediated by protein kinase A (PKA)
and cyclic AMP (cAMP)-dependent pathways stabilizes the protein.
It is thought that this is one mechanism that regulates its nocturnal
peak protein levels (Ganguly et al., 2002) (Fig. 3B). Recent new
evidence has now shown that AANAT translation is also activated
by hnRNP Q, which interacts with an IRES in the Aanat 5⬘-UTR
(Kim et al., 2007a). The expression of hnRNP Q is rhythmic and
its expression profile coincides with the phase of AANAT
expression (Kim et al., 2005). Additionally, hnRNP Q, together
with hnRNP L and hnRNP R, interacts with the 3⬘-UTR of Aanat
mRNA and promotes the degradation of the Aanat transcript (Kim
et al., 2005) (Fig. 3B). Although the expression of these hnRNP
proteins is high when Aanat mRNA expression is relatively low,
mathematical modeling predicts that hnRNP-mediated Aanat
degradation affects the phase and amplitude of Aanat mRNA
fluctuation (Kim et al., 2005). Taken together, these posttranscriptional regulatory mechanisms, in conjunction with the
resulting post-translational modifications, shape the extreme
AANAT expression pattern.
The circadian clock also exerts control over other posttranscriptional events in animals, such as splicing (Belanger et al.,
2006), polyadenylation (Cagampang et al., 1994; Robinson et al.,
1988), deadenylation (Baggs and Green, 2003; Garbarino-Pico et
al., 2007) and translation (Fujimoto et al., 2006; Kim et al., 2002;
Nishii et al., 2006; Yamamoto et al., 2005), although the mechanistic
details of many of these events have not been fully elucidated.
316
Journal of Cell Science 124 (3)
Box 1. Glossary of terms used in circadian biology.
•Amplitude. The variation between the peak and trough of the
oscillation.
•Circadian clock. Internal timing mechanism that coordinates
physiological events with the external environment.
•Free running. Self-sustained rhythms not influenced by
external cues.
•Pacemaker. An oscillator that coordinates the timing of other
oscillators.
•Period. The time for one complete cycle.
•Phase. The time of a point in the rhythm (usually the peak or
trough) with reference to some external point, such as local
time or the onset of activity.
•Suprachiasmatic nucleus (SCN). Area within the
hypothalamus that receives light input from the optic nerve
and is responsible for synchronizing the clocks in peripheral
tissues.
•Temperature compensation. Retaining relatively constant
Journal of Cell Science
periodicity over a broad physiological range of temperatures.
Temperature-induced alternative splicing
Most organisms experience significant variations in ambient
temperature, but the free-running periods (Box 1) of circadian
rhythms are precise and efficiently temperature compensated (Box
1) within a physiological range, even though all biochemical
processes are generally temperature dependent. However,
temperature shifts can often result in the resetting of circadian
clocks, that is they can affect the phase of the circadian oscillation,
similar to the effect of light. Comparable to light provoking several
different post-transcriptional events, cold temperatures induce the
alternative splicing of Per and frq in fly and fungi, respectively,
and, as a result, two different protein isoforms are produced (Colot
et al., 2005; Diernfellner et al., 2005; Majercak et al., 1999) (Fig.
4A,B). The ratio between one isoform and the other does not
appear to be important for temperature compensation, as strains
that possess only one isoform can still maintain consistent rhythms
(Diernfellner et al., 2007; Majercak et al., 1999). Rather, the ratio
of the two isoforms is crucial to robust free-running rhythmicity in
fungi and the phase of Per and PER expression in the fly, providing
a means to fine-tune the clock system in response to ambient
temperature (Diernfellner et al., 2007; Diernfellner et al., 2005;
Majercak et al., 1999). The molecular mechanism underlying this
splicing is unknown, but one hypothesis is that trans-factors that
regulate splicing are also under temperature control. In plants, the
RBPs AtGRP7 and AtGRP8 are known to directly trigger splicing
events. When AtGRP7 or AtGRP8 proteins reach a certain level,
they bind to both of their pre-mRNAs and promote unproductive
splicing coupled to degradation through the nonsense-mediated
decay pathway (Schoning et al., 2008; Staiger et al., 2003), leading
to the decrease in their mRNA level (Fig. 4C). AtGRP7 and
AtGRP8 show robust rhythmic expression at both the mRNA and
protein level, and their rhythmic expression is therefore regulated
by a negative feedback loop of their own. This feedback might also
contribute to the observed delay (approximately 4 hours) between
mRNA and protein expression. Interestingly, mRNA expression of
AtGRP7 and AtGRP8 is induced by cold temperature (Carpenter
et al., 1994), although the protein level of AtGRP7, but not AtGRP8,
is increased (Schoning et al., 2008). Because the splicing of
AtGRP7 and AtGRP8 is regulated by their own protein level, it is
likely that cold temperature also affects the splicing of these
proteins, which could lead to mRNA degradation. Therefore, this
cold-temperature-induced alternative splicing seems to be conserved
at least in plants, Drosophila and Neurospora, and might be a
common mechanism to adapt to cold temperature in other
organisms as well.
The role of miRNAs in the circadian clock
The discovery of miRNAs as regulators of gene expression has
sparked increased interest in determining the genes that are
regulated through these mechanisms and how they relate to
maintaining circadian timing. miRNAs are short (19–25
nucleotides) non-coding RNAs (Shyu et al., 2008) that arise from
primary transcribed miRNAs (pri-miRNAs), which are capped,
adenylated and spliced just like protein-coding mRNAs. Afterwards,
they are cleaved in the nucleus by the RNaseIII endonucleases
Drosha and Pasha, resulting in a shorter (up to 65 nucleotides)
precursor miRNA (pre-miRNA). These pre-miRNAs are then
exported into the cytoplasm, where they are digested further by
Dicer to form a short duplex RNA. This duplex RNA is
incorporated into the functional miRNA-induced silencing complex
(miRISC), which recognizes and processes target mRNAs (Carthew
and Sontheimer, 2009; Chekulaeva and Filipowicz, 2009). miRNAs
recognize and bind to their targets through specific sequences
typically found in their 3⬘-UTRs. One requirement to achieve
binding specificity is an almost perfect match between the 5⬘proximal ‘seed’ region (position 2–8) of the miRNA and its target
mRNA (Carthew and Sontheimer, 2009; Mendes et al., 2009).
Once the miRNA recognizes and interacts with its target mRNA,
protein synthesis is inhibited or deadenylation and degradation are
triggered, but the detailed molecular mechanisms are still being
debated (Carthew and Sontheimer, 2009; Chekulaeva and
Filipowicz, 2009). Several algorithms have been developed that
predict the interaction between miRNAs and target mRNAs, which
are heavily based on the conservation of the seed region (Mendes
et al., 2009). Using this information, several core clock genes, such
as Period1, 2, 3 and both mammalian and Drosophila Clock, have
been predicted to be miRNA targets (Kadener et al., 2009;
Kiriakidou et al., 2004; Meng et al., 2006; Nagel et al., 2009; Yang
et al., 2008). In addition, the effect of miR-141 on mammalian
Clock was validated experimentally (Kiriakidou et al., 2004; Meng
et al., 2006).
miRNAs have been found to target more than 30% of all proteincoding mRNAs; therefore, most, if not all, biological processes
appear to be regulated by miRNAs to at least some degree
(Chekulaeva and Filipowicz, 2009; Croce, 2009). Contributions of
circadian clock function to miRNA expression or vice versa have
been shown in several organisms. For example, expression of some
miRNAs is found to be rhythmic in mouse retina, mouse SCN,
Drosophila brain and Arabidopsis (Cheng et al., 2007; Sire et al.,
2009; Xu et al., 2007; Yang et al., 2008). Other miRNAs are more
directly involved in the clock system, and regulate the circadian
period or response to light (Cheng et al., 2007; Jung et al., 2007;
Kadener et al., 2009). For example, the expression of miR-132 is
induced by a mitogen-activated protein kinase (MAPK)- and cAMP
response element binding (CREB)-dependent mechanism in
response to light, and facilitates the inhibition of a light-induced
phase delay (Cheng et al., 2007). By contrast, the expression of
miR-219 is driven by CLOCK–BMAL1 and its knockdown in
vivo leads to a longer circadian period (Cheng et al., 2007). Another
interesting miRNA with a role in the circadian clock is miR-122,
Post-transcriptional control of circadian rhythms
A
sFRQ
B
l-FRQ
l-FRQ
l-FRQ
l-FRQ
Exon I
sFRQ
Exon II
Exon III
l-FRQ
High temperature
Exon I Exon II Exon III
High temperature
ATG
Exon I
ATG
Exon II
Exon I
Exon II
Exon III
Low temperature
Exon III
Exon I
Exon II Exon III
Low temperature
Exon II Exon III
Advanced Per and PER accumulation
sFRQ
sFRQ
sFRQ
sFRQ
sFRQ
sFRQ
l-FRQ
Advanced evening activity
Journal of Cell Science
l-FRQ
Low temperature
C
Exon I
Exon I
Exon II
Exon III
Exon II
AtGRP7
AtGRP8
Exon III
Exon I
Exon II
Exon III
AtGRP7
Unproductive splicing
AtGRP7
AtGRP7
317
AtGRP7
Exon I
Exon II
Exon III
Degradation
a hepatocyte-specific miRNA. The transcription of miR-122 is
rhythmic in mouse liver only at the pre-miRNA level, whereas the
level of mature miRNA remains constant (Gatfield et al., 2009;
Kojima et al., 2010). Knockdown of miR-122 attenuates the
expression of a disproportionately high number of rhythmic genes
(Gatfield et al., 2009). One of the target mRNAs of miR-122 is the
circadian deadenylase Nocturnin (Kojima et al., 2010), which is a
presumed key factor in circadian post-transcriptional control.
Nocturnin expression is highly rhythmic and is thought to regulate
the clock output pathway, rather than the core clock, most likely
by deadenylating target mRNAs, resulting in changes in their
stability or ability to be translated (Baggs and Green, 2003;
Garbarino-Pico et al., 2007; Green and Besharse, 1996; Green et
al., 2007; Wang et al., 2001).
Concluding remarks
To maintain approximate 24-hour cycles at the molecular level,
clocks must be tightly regulated at several steps to maintain the
correct period, phase and amplitude of the rhythms of thousands
of proteins that generate the wide range of rhythmic biological
processes. As described above, there is now abundant evidence
Fig. 4. Temperature-regulated
alternative splicing of clock genes.
(A)Temperature-sensitive splicing
of frq. At low temperature, splicing
at the non-canonical splice sites
becomes dominant, leading to
higher levels of expression of shortFRQ (sFRQ). At higher temperature,
the canonical splicing site
dominates, leading to higher levels
of long-FRQ (l-FRQ) expression.
(B)Temperature-sensitive splicing
of Drosophila Per. There are two
transcript forms of Per; one includes
an intron sequence in the 3⬘-UTR
and the other does not. Even though
this difference does not alter the
protein structure of PER, this
splicing promotes the earlier
accumulation of Per and PER, and
advances the evening locomotor
activity of fly. This might be a
mechanism for adjustment to winter
time, in which the temperature is
lower and the photoperiod is shorter.
(C)Autoregulation of AtGRP7 and
AtGRP8 expression. AtGRP7 and
AtGRP8 mRNAs are both induced
by cold temperature. The protein
level of AtGRP7, but not AtGRP8,
is also increased by the cold
temperature. Both AtGRP7 and
AtGRP8 interact with their premRNA, and promote the splicing
that yields aberrant mRNA. As a
consequence, these abnormal RNAs
are degraded rapidly in the cell.
that post-transcriptional mechanisms play an important role in
shaping these rhythms. However, much of this information is only
anecdotal and additional studies need to be performed to provide
a detailed knowledge of these regulatory mechanisms. A list of the
publications demonstrating post-transcriptional regulation in the
circadian field is presented in Table 1.
For mRNAs to be rhythmic, their half-lives must be relatively
short and several reports have shown that the stability of these
cycling mRNAs can change throughout the 24-hour period.
However, it is not clear how broad this circadian change in
mRNA stability is among cycling genes; are all cycling genes
regulated in such a way or does this only occur in a subset?
Furthermore, the question remains how the degradation of
mRNAs is governed in a circadian-dependent manner, particularly
as these rhythmic mRNAs peak at different times of the day. The
recent work that demonstrates a direct role for the exosome in
circadian control in Neurospora is highly exciting, because if this
aspect of exosome function is conserved in other organisms, it
could constitute a general mechanism for the rhythmic regulation
of the stability of target mRNAs. Because hnRNPs can recruit the
exosome to target mRNAs for their degradation (Hessle et al.,
318
Journal of Cell Science 124 (3)
Table 1. List of circadian-related genes regulated by post-transcriptional mechanisms
Post-transcriptional
mechanism
Translation
Journal of Cell Science
mRNA stability
Alternative splicing
miRNA regulation
Gene name
Period 1
Organism
Mus musculus
Period 2
M. musculus
Rev-erbα
M. musculus
Aanat
Rattus norvegicus
Period
Eip74EF
D. melanogaster
D. melanogaster
WC-1
N. crassa
CCA1
A. thaliana
Lbp
Lingulodinium
Period 1
M. musculus
Period 2
M. musculus
Period 3
Cryptochrome 1
M. musculus
M. musculus
Arginine
Vasopressin
Aanat
M. musculus
R. norvegicus
Period
Clock
Frequency
CCR-Like
SEN1
Presenilin-2
D. melanogaster
D. melanogaster
N. crassa
A. thaliana
A. thaliana
M. musculus
Period
D. melanogaster
Frequency
N. crassa
Atgrp7
Atgrp8
miR-192/194
miR-141
miR-132
A. thaliana
A. thaliana
Homo sapiens
H. sapiens
M. musculus
miR-219
miR-122
M. musculus
M. musculus
bantam
D. melanogaster
miR-263a/b
As-frequency
D. melanogaster
N. crassa
miR-172
A. thaliana
Regulation
3 -UTR-mediated upregulated translation through
interactions with LARK
Rhythmic translation from a non-rhythmic transcript
IRES-dependent translation through interactions with
hnRNP I and hnRNP Q
Rhythmic IRES-dependent translation through
hnRNP Q interactions
3 -UTR-mediated rhythmic translation
Upregulated translation through interactions with
LARK
Increases in WC-1 expression correlated with FRQ
expression
Rhythmic protein expression from a non-rhythmic
transcript
3 -UTR-mediated rhythmic translation through
CCTR interactions
3 -UTR-mediated mRNA degradation
3 -UTR-mediated mRNA decay through hnRNP I
interactions
3 -UTR-mediated mRNA degradation
3 -UTR-mediated degradation through hnRNP D
interactions
Rhythmic changes in poly(A) tail length
Rhythmic mRNA degradation through hnRNP Q,
hnRNP R, and hnRNP L interactions
Rhythmic changes in mRNA stability
Rhythmic changes in mRNA stability
mRNA stability through interactions with FFC
mRNA degradation through the dst element pathway
mRNA degradation through the dst element pathway
Rhythmic expression of two alternatively spliced
transcripts
Thermosensitive splicing of an intron in the 3 -UTR
Thermosensitive splicing generating two isoforms
Autoregulated expression through alternative splicing
Autoregulated expression through alternative splicing
Decreases PER 1, 2, and 3 translation
Decreases CLOCK translation
Inhibition of a light-induced phase delay by
unknown targets
Regulation of circadian period by unknown targets
Alterations of rhythmically expressed genes in the
liver
Regulation of period length possibly though clk
expression
Rhythmically expressed miRNAs
Rhythmically expressed antisense RNA modulating
frq rhythmicity
Regulation of photoperiodic flowering by controlling
the amplitude of FLOWERING LOCUS
expression
2009), it is tempting to speculate that rhythmically expressed
RBPs might enable phase-dependent recruitment of the exosome
in a target-specific manner. This possibility has not yet been
widely explored, but is an area ripe for further investigation.
Another interesting aspect of many cycling genes is the time lag
(typically 4–6 hours) that often occurs between mRNA and protein
expression, suggesting regulation at the translational level. It is
not known whether this lag is caused by a universal circadian
regulatory system or is the result of the functions of different
RNA-specific mechanisms.
References
(Kojima et al., 2007)
(Fujimoto et al., 2006; Nishii et al.,
2006; Yamamoto et al., 2005)
(Kim et al., 2010)
(Kim et al., 2007a)
(Chen et al., 1998)
(Huang et al., 2007)
(Lee et al., 2000)
(Yakir et al., 2007)
(Mittag et al., 1994)
(Wilsbacher et al., 2002; Kojima et
al., 2003)
(Woo et al., 2009)
(Kwak et al., 2006)
(Woo et al., 2010)
(Cagampang et al., 1994; Robinson
et al., 1988)
(Kim et al., 2005)
(So and Rosbash, 1997)
(Kim et al., 2002)
(Guo et al., 2009)
(Lidder et al., 2005)
(Lidder et al., 2005)
(Belanger et al., 2006)
(Majercak et al., 1999; Majercak et
al., 2004)
(Colot et al., 2005; Diernfellner et
al., 2005; Diernfellner et al., 2007)
(Schoning et al., 2008)
(Staiger et al., 2003)
(Nagel et al., 2009)
(Meng et al., 2006)
(Cheng et al., 2007)
(Cheng et al., 2007)
(Gatfield et al., 2009; Kojima et al.,
2010)
(Kadener et al., 2009)
(Yang et al., 2008)
(Kramer et al., 2003)
(Jung et al., 2007)
Research over the past two decades has generated a growing
appreciation of the importance of gene regulatory processes at the
post-transcriptional level. It has become very clear that both
transcriptional and post-transcriptional processes are necessary to
generate robust circadian rhythms of mRNA expression. Despite
this realization, our understanding of circadian post-transcriptional
mechanisms lags far behind our understanding of clock regulation
at the transcriptional level. This is partially owing to the lack of
well-developed methodologies to find post-transcriptionally
regulated genes on a large scale. The development of such methods
Post-transcriptional control of circadian rhythms
is likely to lead to the discovery of many more genes and
mechanisms that are under post-transcriptional control.
Financial support for this work was provided by NIH grant
GM076626 to C.B.G. Deposited in PMC for release after 12 months.
Journal of Cell Science
References
Baggs, J. E. and Green, C. B. (2003). Nocturnin, a deadenylase in Xenopus laevis retina:
a mechanism for posttranscriptional control of circadian-related mRNA. Curr. Biol. 13,
189-198.
Bargiello, T. A. and Young, M. W. (1984). Molecular genetics of a biological clock in
Drosophila. Proc. Natl. Acad. Sci. USA 81, 2142-2146.
Belanger, V., Picard, N. and Cermakian, N. (2006). The circadian regulation of Presenilin2 gene expression. Chronobiol. Int. 23, 747-766.
Belostotsky, D. (2009). Exosome complex and pervasive transcription in eukaryotic
genomes. Curr. Opin. Cell Biol. 21, 352-358.
Brunner, M. and Kaldi, K. (2008). Interlocked feedback loops of the circadian clock of
Neurospora crassa. Mol. Microbiol. 68, 255-262.
Cagampang, F. R., Yang, J., Nakayama, Y., Fukuhara, C. and Inouye, S. T. (1994).
Circadian variation of arginine-vasopressin messenger RNA in the rat suprachiasmatic
nucleus. Brain Res. Mol. Brain Res. 24, 179-184.
Carpenter, C. D., Kreps, J. A. and Simon, A. E. (1994). Genes encoding glycine-rich
Arabidopsis thaliana proteins with RNA-binding motifs are influenced by cold treatment
and an endogenous circadian rhythm. Plant Physiol. 104, 1015-1025.
Carthew, R. W. and Sontheimer, E. J. (2009). Origins and mechanisms of miRNAs and
siRNAs. Cell 136, 642-655.
Chaudhury, A., Chander, P. and Howe, P. H. (2010). Heterogeneous nuclear
ribonucleoproteins (hnRNPs) in cellular processes: focus on hnRNP E1’s multifunctional
regulatory roles. RNA 16, 1449-1462.
Chekulaeva, M. and Filipowicz, W. (2009). Mechanisms of miRNA-mediated posttranscriptional regulation in animal cells. Curr. Opin. Cell Biol. 21, 452-460.
Chen, M., Chory, J. and Fankhauser, C. (2004). Light signal transduction in higher
plants. Annu. Rev. Genet. 38, 87-117.
Chen, Y., Hunter-Ensor, M., Schotland, P. and Sehgal, A. (1998). Alterations of per
RNA in noncoding regions affect periodicity of circadian behavioral rhythms. J. Biol.
Rhythms 13, 364-379.
Cheng, H. Y., Papp, J. W., Varlamova, O., Dziema, H., Russell, B., Curfman, J. P.,
Nakazawa, T., Shimizu, K., Okamura, H., Impey, S. et al. (2007). microRNA
modulation of circadian-clock period and entrainment. Neuron 54, 813-829.
Colot, H. V., Loros, J. J. and Dunlap, J. C. (2005). Temperature-modulated alternative
splicing and promoter use in the Circadian clock gene frequency. Mol. Biol. Cell 16,
5563-5571.
Croce, C. M. (2009). Causes and consequences of microRNA dysregulation in cancer.
Nat. Rev. Genet. 10, 704-714.
Diernfellner, A., Colot, H. V., Dintsis, O., Loros, J. J., Dunlap, J. C. and Brunner, M.
(2007). Long and short isoforms of Neurospora clock protein FRQ support temperaturecompensated circadian rhythms. FEBS Lett. 581, 5759-5764.
Diernfellner, A. C., Schafmeier, T., Merrow, M. W. and Brunner, M. (2005). Molecular
mechanism of temperature sensing by the circadian clock of Neurospora crassa. Genes
Dev. 19, 1968-1973.
Duffield, G. E. (2003). DNA microarray analyses of circadian timing: the genomic basis
of biological time. J. Neuroendocrinol. 15, 991-1002.
Fujimoto, Y., Yagita, K. and Okamura, H. (2006). Does mPER2 protein oscillate
without its coding mRNA cycling? Post-transcriptional regulation by cell clock. Genes
Cells 11, 525-530.
Ganguly, S., Coon, S. L. and Klein, D. C. (2002). Control of melatonin synthesis in the
mammalian pineal gland: the critical role of serotonin acetylation. Cell Tissue Res. 309,
127-137.
Garbarino-Pico, E., Niu, S., Rollag, M. D., Strayer, C. A., Besharse, J. C. and Green,
C. B. (2007). Immediate early response of the circadian polyA ribonuclease nocturnin
to two extracellular stimuli. RNA 13, 745-755.
Garceau, N. Y., Liu, Y., Loros, J. J. and Dunlap, J. C. (1997). Alternative initiation of
translation and time-specific phosphorylation yield multiple forms of the essential clock
protein FREQUENCY. Cell 89, 469-476.
Gatfield, D., Le Martelot, G., Vejnar, C. E., Gerlach, D., Schaad, O., Fleury-Olela, F.,
Ruskeepaa, A. L., Oresic, M., Esau, C. C., Zdobnov, E. M. et al. (2009). Integration
of microRNA miR-122 in hepatic circadian gene expression. Genes Dev. 23, 13131326.
Green, C. B. and Besharse, J. C. (1996). Identification of a novel vertebrate circadian
clock-regulated gene encoding the protein nocturnin. Proc. Natl. Acad. Sci. USA 93,
14884-14888.
Green, C. B., Douris, N., Kojima, S., Strayer, C. A., Fogerty, J., Lourim, D., Keller,
S. R. and Besharse, J. C. (2007). Loss of Nocturnin, a circadian deadenylase, confers
resistance to hepatic steatosis and diet-induced obesity. Proc. Natl. Acad. Sci. USA 104,
9888-9893.
Guo, J., Cheng, P., Yuan, H. and Liu, Y. (2009). The exosome regulates circadian gene
expression in a posttranscriptional negative feedback loop. Cell 138, 1236-1246.
Gutierrez, R. A., Ewing, R. M., Cherry, J. M. and Green, P. J. (2002). Identification
of unstable transcripts in Arabidopsis by cDNA microarray analysis: rapid decay is
associated with a group of touch- and specific clock-controlled genes. Proc. Natl. Acad.
Sci. USA 99, 11513-11518.
319
Hastings, J. W. (2007). The Gonyaulax clock at 50, translational control of circadian
expression. Cold Spring Harb. Symp. Quant. Biol. 72, 141-144.
Hastings, M. H., Maywood, E. S. and O’Neill, J. S. (2008). Cellular circadian pacemaking
and the role of cytosolic rhythms. Curr. Biol. 18, R805-R815.
Hessle, V., Bjork, P., Sokolowski, M., Gonzalez de Valdivia, E., Silverstein, R.,
Artemenko, K., Tyagi, A., Maddalo, G., Ilag, L., Helbig, R. et al. (2009). The
exosome associates cotranscriptionally with the nascent pre-mRNP through interactions
with heterogeneous nuclear ribonucleoproteins. Mol. Biol. Cell 20, 3459-3470.
Hock, J., Weinmann, L., Ender, C., Rudel, S., Kremmer, E., Raabe, M., Urlaub, H.
and Meister, G. (2007). Proteomic and functional analysis of Argonaute-containing
mRNA-protein complexes in human cells. EMBO Rep. 8, 1052-1060.
Huang, Y., Genova, G., Roberts, M. and Jackson, F. R. (2007). The LARK RNAbinding protein selectively regulates the circadian eclosion rhythm by controlling E74
protein expression. PLoS ONE 2, e1107.
Huang, Y., Howlett, E., Stern, M. and Jackson, F. R. (2009). Altered LARK expression
perturbs development and physiology of the Drosophila PDF clock neurons. Mol. Cell
Neurosci. 41, 196-205.
Iuvone, P. M., Tosini, G., Pozdeyev, N., Haque, R., Klein, D. C. and Chaurasia, S. S.
(2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and
melatonin in the retina. Prog. Retin. Eye Res. 24, 433-456.
Johnson, C. H., Roeber, J. F. and Hastings, J. W. (1984). Circadian changes in enzyme
concentration account for rhythm of enzyme activity in gonyaulax. Science 223, 14281430.
Jung, J. H., Seo, Y. H., Seo, P. J., Reyes, J. L., Yun, J., Chua, N. H. and Park, C. M.
(2007). The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering
independent of CONSTANS in Arabidopsis. Plant Cell 19, 2736-2748.
Kadener, S., Menet, J. S., Sugino, K., Horwich, M. D., Weissbein, U., Nawathean, P.,
Vagin, V. V., Zamore, P. D., Nelson, S. B. and Rosbash, M. (2009). A role for
microRNAs in the Drosophila circadian clock. Genes Dev. 23, 2179-2191.
Keene, J. D. (2010). Minireview: global regulation and dynamics of ribonucleic acid.
Endocrinology 151, 1391-1397.
Kim, D. Y., Woo, K. C., Lee, K. H., Kim, T. D. and Kim, K. T. (2010). hnRNP Q and
PTB modulate the circadian oscillation of mouse Rev-erb {alpha} via IRES-mediated
translation. Nucleic Acids Res. 38, 7068-7078.
Kim, E. Y., Bae, K., Ng, F. S., Glossop, N. R., Hardin, P. E. and Edery, I. (2002).
Drosophila CLOCK protein is under posttranscriptional control and influences lightinduced activity. Neuron 34, 69-81.
Kim, J. Y., Song, H. R., Taylor, B. L. and Carre, I. A. (2003). Light-regulated translation
mediates gated induction of the Arabidopsis clock protein LHY. EMBO J. 22, 935-944.
Kim, T. D., Kim, J. S., Kim, J. H., Myung, J., Chae, H. D., Woo, K. C., Jang, S. K.,
Koh, D. S. and Kim, K. T. (2005). Rhythmic serotonin N-acetyltransferase mRNA
degradation is essential for the maintenance of its circadian oscillation. Mol. Cell. Biol.
25, 3232-3246.
Kim, T. D., Woo, K. C., Cho, S., Ha, D. C., Jang, S. K. and Kim, K. T. (2007a).
Rhythmic control of AANAT translation by hnRNP Q in circadian melatonin production.
Genes Dev. 21, 797-810.
Kim, W. Y., Fujiwara, S., Suh, S. S., Kim, J., Kim, Y., Han, L., David, K., Putterill,
J., Nam, H. G. and Somers, D. E. (2007b). ZEITLUPE is a circadian photoreceptor
stabilized by GIGANTEA in blue light. Nature 449, 356-360.
Kiriakidou, M., Nelson, P. T., Kouranov, A., Fitziev, P., Bouyioukos, C., Mourelatos,
Z. and Hatzigeorgiou, A. (2004). A combined computational-experimental approach
predicts human microRNA targets. Genes Dev. 18, 1165-1178.
Kojima, S., Hirose, M., Tokunaga, K., Sakaki, Y. and Tei, H. (2003). Structural and
functional analysis of 3⬘ untranslated region of mouse Period1 mRNA. Biochem.
Biophys. Res. Commun. 301, 1-7.
Kojima, S., Matsumoto, K., Hirose, M., Shimada, M., Nagano, M., Shigeyoshi, Y.,
Hoshino, S., Ui-Tei, K., Saigo, K., Green, C. B. et al. (2007). LARK activates
posttranscriptional expression of an essential mammalian clock protein, PERIOD1.
Proc. Natl. Acad. Sci. USA 104, 1859-1864.
Kojima, S., Gatfield, D., Esau, C. C. and Green, C. B. (2010). MicroRNA-122 modulates
the rhythmic expression profile of the circadian deadenylase Nocturnin in mouse liver.
PLoS ONE 5, e11264.
Konopka, R. J. and Benzer, S. (1971). Clock mutants of Drosophila melanogaster. Proc.
Natl. Acad. Sci. USA 68, 2112-2116.
Kramer, C., Loros, J. J., Dunlap, J. C. and Crosthwaite, S. K. (2003). Role for
antisense RNA in regulating circadian clock function in Neurospora crassa. Nature 421,
948-952.
Kwak, E., Kim, T. D. and Kim, K. T. (2006). Essential role of 3⬘-untranslated regionmediated mRNA decay in circadian oscillations of mouse Period3 mRNA. J. Biol.
Chem. 281, 19100-19106.
Lai, M. C., Kuo, H. W., Chang, W. C. and Tarn, W. Y. (2003). A novel splicing regulator
shares a nuclear import pathway with SR proteins. EMBO J. 22, 1359-1369.
Lee, K., Loros, J. J. and Dunlap, J. C. (2000). Interconnected feedback loops in the
Neurospora circadian system. Science 289, 107-110.
Lidder, P., Gutierrez, R. A., Salome, P. A., McClung, C. R. and Green, P. J. (2005).
Circadian control of messenger RNA stability. Association with a sequence-specific
messenger RNA decay pathway. Plant Physiol. 138, 2374-2385.
Lin, J. C., Hsu, M. and Tarn, W. Y. (2007). Cell stress modulates the function of splicing
regulatory protein RBM4 in translation control. Proc. Natl. Acad. Sci. USA 104, 22352240.
Majercak, J., Chen, W. F. and Edery, I. (2004). Splicing of the period gene 3¢-terminal
intron is regulated by light, circadian clock factors, and phospholipase C. Mol. Cell.
Biol. 24, 3359-3372.
Journal of Cell Science
320
Journal of Cell Science 124 (3)
Majercak, J., Sidote, D., Hardin, P. E. and Edery, I. (1999). How a circadian clock
adapts to seasonal decreases in temperature and day length. Neuron 24, 219-230.
Martinez-Garcia, J. F., Huq, E. and Quail, P. H. (2000). Direct targeting of light signals
to a promoter element-bound transcription factor. Science 288, 859-863.
McNeil, G. P., Zhang, X., Genova, G. and Jackson, F. R. (1998). A molecular rhythm
mediating circadian clock output in Drosophila. Neuron 20, 297-303.
Mendes, N. D., Freitas, A. T. and Sagot, M. F. (2009). Current tools for the identification
of miRNA genes and their targets. Nucleic Acids Res. 37, 2419-2433.
Meng, F., Henson, R., Lang, M., Wehbe, H., Maheshwari, S., Mendell, J. T., Jiang, J.,
Schmittgen, T. D. and Patel, T. (2006). Involvement of human micro-RNA in growth
and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology
130, 2113-2129.
Mittag, M. (1996). Conserved circadian elements in phylogenetically diverse algae. Proc.
Natl. Acad. Sci. USA 93, 14401-14404.
Mittag, M., Lee, D. H. and Hastings, J. W. (1994). Circadian expression of the luciferinbinding protein correlates with the binding of a protein to the 3⬘ untranslated region of
its mRNA. Proc. Natl. Acad. Sci. USA 91, 5257-5261.
Morse, D., Milos, P. M., Roux, E. and Hastings, J. W. (1989). Circadian regulation of
bioluminescence in Gonyaulax involves translational control. Proc. Natl. Acad. Sci.
USA 86, 172-176.
Nagel, R., Clijsters, L. and Agami, R. (2009). The miRNA-192/194 cluster regulates the
Period gene family and the circadian clock. FEBS J. 276, 5447-5455.
Newby, L. M. and Jackson, F. R. (1993). A new biological rhythm mutant of Drosophila
melanogaster that identifies a gene with an essential embryonic function. Genetics 135,
1077-1090.
Newby, L. M. and Jackson, F. R. (1996). Regulation of a specific circadian clock output
pathway by lark, a putative RNA-binding protein with repressor activity. J. Neurobiol.
31, 117-128.
Nishii, K., Yamanaka, I., Yasuda, M., Kiyohara, Y. B., Kitayama, Y., Kondo, T. and
Yagita, K. (2006). Rhythmic post-transcriptional regulation of the circadian clock
protein mPER2 in mammalian cells: a real-time analysis. Neurosci. Lett. 401, 44-48.
Perez-Amador, M. A., Lidder, P., Johnson, M. A., Landgraf, J., Wisman, E. and
Green, P. J. (2001). New molecular phenotypes in the dst mutants of Arabidopsis
revealed by DNA microarray analysis. Plant Cell 13, 2703-2717.
Pruneda-Paz, J. L. and Kay, S. A. (2010). An expanding universe of circadian networks
in higher plants. Trends Plant Sci. 15, 259-265.
Raineri, I., Wegmueller, D., Gross, B., Certa, U. and Moroni, C. (2004). Roles of AUF1
isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA
interference. Nucleic Acids Res. 32, 1279-1288.
Reddy, A. B., Karp, N. A., Maywood, E. S., Sage, E. A., Deery, M., O’Neill, J. S.,
Wong, G. K., Chesham, J., Odell, M., Lilley, K. S. et al. (2006). Circadian orchestration
of the hepatic proteome. Curr. Biol. 16, 1107-1115.
Reddy, P., Zehring, W. A., Wheeler, D. A., Pirrotta, V., Hadfield, C., Hall, J. C. and
Rosbash, M. (1984). Molecular analysis of the period locus in Drosophila melanogaster
and identification of a transcript involved in biological rhythms. Cell 38, 701-710.
Robinson, B. G., Frim, D. M., Schwartz, W. J. and Majzoub, J. A. (1988). Vasopressin
mRNA in the suprachiasmatic nuclei: daily regulation of polyadenylate tail length.
Science 241, 342-344.
Roseboom, P. H., Coon, S. L., Baler, R., McCune, S. K., Weller, J. L. and Klein, D. C.
(1996). Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in
serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland.
Endocrinology 137, 3033-3045.
Sawicka, K., Bushell, M., Spriggs, K. A. and Willis, A. E. (2008). Polypyrimidine-tractbinding protein: a multifunctional RNA-binding protein. Biochem. Soc. Trans. 36, 641647.
Schoning, J. C., Streitner, C., Meyer, I. M., Gao, Y. and Staiger, D. (2008). Reciprocal
regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop
coupling alternative splicing to nonsense-mediated decay in Arabidopsis. Nucleic Acids
Res. 36, 6977-6987.
Schroeder, A. J., Genova, G. K., Roberts, M. A., Kleyner, Y., Suh, J. and Jackson, F.
R. (2003). Cell-specific expression of the lark RNA-binding protein in Drosophila
results in morphological and circadian behavioral phenotypes. J. Neurogenet. 17, 139169.
Shyu, A. B., Wilkinson, M. F. and van Hoof, A. (2008). Messenger RNA regulation: to
translate or to degrade. EMBO J. 27, 471-481.
Singh, R., Valcarcel, J. and Green, M. R. (1995). Distinct binding specificities and
functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268,
1173-1176.
Sire, C., Moreno, A. B., Garcia-Chapa, M., Lopez-Moya, J. J. and San Segundo, B.
(2009). Diurnal oscillation in the accumulation of Arabidopsis microRNAs, miR167,
miR168, miR171 and miR398. FEBS Lett. 583, 1039-1044.
So, W. V. and Rosbash, M. (1997). Post-transcriptional regulation contributes to Drosophila
clock gene mRNA cycling. EMBO J. 16, 7146-7155.
Staiger, D., Zecca, L., Wieczorek Kirk, D. A., Apel, K. and Eckstein, L. (2003).
The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its
expression by influencing alternative splicing of its own pre-mRNA. Plant J. 33,
361-371.
Sun, Z. S., Albrecht, U., Zhuchenko, O., Bailey, J., Eichele, G. and Lee, C. C. (1997).
RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell 90, 10031011.
Tei, H., Okamura, H., Shigeyoshi, Y., Fukuhara, C., Ozawa, R., Hirose, M. and
Sakaki, Y. (1997). Circadian oscillation of a mammalian homologue of the Drosophila
period gene. Nature 389, 512-516.
Wang, Y., Osterbur, D. L., Megaw, P. L., Tosini, G., Fukuhara, C., Green, C. B. and
Besharse, J. C. (2001). Rhythmic expression of Nocturnin mRNA in multiple tissues
of the mouse. BMC Dev. Biol. 1, 9.
Wang, Z. Y. and Tobin, E. M. (1998). Constitutive expression of the CIRCADIAN
CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its
own expression. Cell 93, 1207-1217.
Wijnen, H. and Young, M. W. (2006). Interplay of circadian clocks and metabolic
rhythms. Annu. Rev. Genet. 40, 409-448.
Wilsbacher, L. D., Yamazaki, S., Herzog, E. D., Song, E. J., Radcliffe, L. A., Abe, M.,
Block, G., Spitznagel, E., Menaker, M. and Takahashi, J. S. (2002). Photic and
circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo. Proc. Natl.
Acad. Sci. USA 99, 489-494.
Woo, K. C., Kim, T. D., Lee, K. H., Kim, D. Y., Kim, W., Lee, K. Y. and Kim, K. T.
(2009). Mouse period 2 mRNA circadian oscillation is modulated by PTB-mediated
rhythmic mRNA degradation. Nucleic Acids Res. 37, 26-37.
Woo, K. C., Ha, D. C., Lee, K. H., Kim, D. Y., Kim, T. D. and Kim, K. T. (2010).
Circadian amplitude of cryptochrome 1 is modulated by mRNA stability regulation via
cytoplasmic hnRNP D oscillation. Mol. Cell. Biol. 30, 197-205.
Wuarin, J., Falvey, E., Lavery, D., Talbot, D., Schmidt, E., Ossipow, V., Fonjallaz, P.
and Schibler, U. (1992). The role of the transcriptional activator protein DBP in
circadian liver gene expression. J. Cell Sci. Suppl. 16, 123-127.
Xu, S., Witmer, P. D., Lumayag, S., Kovacs, B. and Valle, D. (2007). MicroRNA
(miRNA) transcriptome of mouse retina and identification of a sensory organ-specific
miRNA cluster. J. Biol. Chem. 282, 25053-25066.
Yakir, E., Hilman, D., Hassidim, M. and Green, R. M. (2007). CIRCADIAN CLOCK
ASSOCIATED1 transcript stability and the entrainment of the circadian clock in
Arabidopsis. Plant Physiol. 145, 925-932.
Yamamoto, Y., Yagita, K. and Okamura, H. (2005). Role of cyclic mPer2 expression in
the mammalian cellular clock. Mol. Cell. Biol. 25, 1912-1921.
Yang, M., Lee, J. E., Padgett, R. W. and Edery, I. (2008). Circadian regulation of a
limited set of conserved microRNAs in Drosophila. BMC Genomics 9, 83.