RESEARCH ARTICLE| NEUROSCIENCE

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Circadian ribosome profiling reveals a role for the Period2
upstream open reading frame in sleep
Arthur Milliusa,b,c , Rikuhiro G. Yamadaa, Hiroshi Fujishimaa, Kazuhiko Maedaband Hiroki R. Uedaa,g,1, Daron M. Standleyc, Kenta Sumiyamad, Dimitri Perrine,f,Downloaded from https://www.pnas.org by UNIVERSITY OF TOKYO MEDICAL LIBRARY on November 9, 2023 from IP address 157.82.128.3.

Edited by Joseph Takahashi, The University of Texas Southwestern Medical Center, Dallas, TX; received August 30, 2022; accepted August 29, 2023

Many mammalian proteins have circadian cycles of production and degradation, andmany of these rhythms are altered posttranscriptionally. We used ribosome profiling to
examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-­h period from circadian-­entrained mice transferred to constantdarkness conditions and by comparing ribosome binding levels to protein levels for 16
circadian proteins. We observed large differences in ribosome binding levels compared
to protein levels, and we observed delays between peak ribosome binding and peakprotein abundance. We found extensive binding of ribosomes to upstream open reading
frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An
increase in the number of uORFs in the 5′UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic
reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescencereporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF
cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced
ribosome binding on Per2, and reduced total sleep time compared to that in wild-­type
mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can
impact physiological rhythms and sleep.

Significance
Period (Per) is an iconic gene in
the field of circadian rhythms
since its discovery in 1971 by
Seymour Benzer and Ronald
Konopka in fruit flies. The
inhibitory feedback loop of PER
protein drives circadian rhythms.
We show that Per2 is regulatedby an upstream open reading
frame (uORF) in the 5′
untranslated region of the Per2
mRNA. Mutation of the Per2
uORF altered the amplitude of
luciferase reporter expression inwell-­characterized cell culture
models. Per2 uORF mutant mice
had significantly elevated Per2mRNA levels and exhibited sleep
loss, particularly during light-­to-­dark and dark-­to-­light transitions,which suggests a role for uORFs
in modulating molecular andphysiological circadian rhythms.

circadian rhythms | ribosome profiling | uORF | mass spectrometry | RNA

Life is remarkably adapted to the 24-­hour rotational movement of the earth. In mammals,
the molecular time-­keeping mechanism for circadian rhythms relies primarily on a hierarchical network of transcription activators and repressors in cells and tissues (1). In thepast, circadian clocks have been measured using systems approaches to quantify
genome-­wide changes in RNA levels (2), which has resulted in an understanding of thetranscriptional regulatory network; however, less is known about how translation andposttranscriptional regulation influence biological rhythms.
About 10% of genes in the liver have circadian oscillations in the steady-­state levels ofmRNA (3), but rhythmic transcription accounts for only some of these rhythms (4). For
example, the timing of when a circadian mRNA is expressed does not necessarily correspond to that of mRNA translation or peak protein abundance (5). Some proteins have24-­h rhythms in abundance in the absence of rhythmic RNA expression (6, 7), which
may suggest a role for rhythmic translation in regulating the clock (8).
In mouse liver, detection of low-­abundant components of the core circadian circuit
using systems proteomics is difficult (8, 9), unless special care is taken to examine a particular protein on a case-­by-­case basis (10) or by using advanced mass spectrometry
techniques (11, 12). Researchers have used next-­generation sequencing of ribosome-­boundmRNA protected from RNAse degradation to understand how translation regulation
affects protein output (13, 14). Previous studies using ribosome profiling to measure daily
rhythms focused on a cell culture model (15) or mouse tissues in light–dark conditions(16–18) to examine rhythms in diurnal gene expression, which may be influenced by
noncircadian time-­keeping systems. These studies also examined the timing between RNA
abundance and ribosome binding, but it remains unclear how circadian translation relates
to peak protein abundance in terms of protein turnover and timing. For example, thepeak abundance of PER and CRY proteins is delayed relative to the expression of theirmRNA in the liver (19) and suprachiasmatic nuclei (SCN) (20). This difference in timing
may result from a delay in RNA processing before translation, a delay during translation,a delay in protein transport from one cellular location to another, or a delay in protein
turnover (21, 22).
Previously, we developed a mass spectrometry method called MS-­based Quantification
By isotope-­labeled Cell-­free products (MS-­QBiC) to determine the absolute protein levelsof 16 selected circadian proteins in mice liver over a 24-­h period (10). This method takes
advantage of the PURE system (23) for reconstituting cell-­free protein expression of
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Author contributions: A.M., R.G.Y., K.S., D.P., and H.R.U.
designed research; A.M., R.G.Y., H.F., and K.S. performed
research; K.M. and D.M.S. contributed new reagents/
analytic tools; A.M. and D.P. analyzed data; and A.M.
wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2023 the Author(s). Published by PNAS.
This open access article is distributed under Creative
Commons Attribution License 4.0 (CC BY).
1

To whom correspondence may be addressed. Email:uedah-­tky@umin.ac.jp.
This article contains supporting information online athttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.​2214636120/-­/DCSupplemental.
Published September 28, 2023.

https://doi.org/10.1073/pnas.2214636120

1 of 9optimal peptide standards for detection and quantification by
selected reaction monitoring-­based targeted proteomics analysis.
We found delays between the peak levels of RNA expression, as
measured by qPCR, and the abundance of the corresponding
protein, suggesting either a delay in posttranscriptional RNA processing or in protein turnover. Here, we investigated the same liver
samples by ribosome profiling in order to understand the timingof ribosome binding compared to peak protein and RNA levels.
We found that upstream open reading frames (uORFs) modulatedtranslation globally, repressed reporter expression in a combinatorial manner, and suppressed expression of Per2. Mutation of thePer2 uORF in mice reduced total sleep duration, particularly
during the early morning and early evening, without disrupting
the circadian period, which suggests that uORF-­mediated repression may impact physiological behaviors.
Results

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Ribosome Profiling of the Liver from Mice in Constant Darkness
Conditions. An experimental workflow was designed to analyze

ribosome-­protected mRNA fragments from liver samples
previously examined by MS-­QBiC (10) (SI Appendix, Fig. S1).
Briefly, mice were entrained to a 12-­h light/12-­h dark (LD) cyclefor 14 d, transferred to constant darkness (DD) for 24 h, and killed
at circadian times (CT0, CT4, CT8, CT12, CT16, CT20, andCT24). Liver samples from two mice were collected and analyzed
following established ribosome profiling protocols (24). We
prepared ribosome profiling libraries and sequenced ~70 million
reads per sample; this resulted in 25 to 45 million reads that could
be mapped to mRNA (25) (SI Appendix, Table S1). Ribosome-­
protected fragments primarily aligned to the coding regions and5′ untranslated regions (5′UTRs) of the mRNA, with few reads
mapping to the 3′ untranslated region (3′UTR) (SI Appendix,
Fig. S2A). Alignment of coding sequence (CDS)-­mapped reads
based on the footprint length revealed reading frame periodicity(SI Appendix, Fig. S2B). Reads were of the expected size, mappedwith a high percentage to mRNA, and were correlated between
samples (SI Appendix, Fig. S2 C–E).
From ~14,000 well-­translated transcripts (defined by a medianof at least two reads per five-­codon mRNA window), we identified
rhythms in ribosome-­protected read fragments using the JTK_CYCLE algorithm (26), yielding 2,952 rhythmic transcripts withan adjusted P < 0.05, including well-­known circadian transcripts
such as Bmal1, Per1, Per2, Clock, and Cry1 (Dataset S1 and
SI Appendix, Table S2 and Fig. S6A).
Relationship between Ribosome Profiling Reads and Protein
Levels. We compared the timing of protein production, as

measured by ribosome profiling reads, to the absolute numberof protein molecules per cell for 16 previously reported corecircadian proteins (10). There was broad agreement in the timingof ribosome binding compared to that of protein abundance(Fig. 1A). However, for several circadian proteins, such as BMAL1
and CLOCK, there was a delay of approximately 6 h between peakribosome binding and peak protein abundance, clearly outside the4-­h range of our sampling intervals (Fig. 1B), which suggested
that there is a posttranslational delay in protein turnover for thesemolecules rather than a posttranscriptional delay in ribosome
binding. We compared the average number of ribosome profilingreads to the number of protein molecules over a 24-­h period but
found that the two values were poorly correlated (Fig. 1C). We
measured this protein production efficiency (as defined by theratio of our mass-­spectrometry protein levels to ribosome profilingreads) at each time point. For some proteins, such as BHLHE40, a
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large amount of ribosome binding resulted in a moderate amountof protein (Fig. 1D), whereas for other proteins, such as PER2, amuch smaller amount of ribosome binding resulted in the sameamount of protein as BHLHE40 (Fig. 1E), which suggests that
BHLHE40 protein is more unstable than PER2 or that someother factor is limiting the amount of BHLHE40 produced. Thus,
ribosome profiling reads can provide an approximate estimate forwhen a protein is produced, but protein abundance reflects bothprotein production and posttranslational mechanisms to control
overall protein levels (27).
When we compared mRNA and protein abundance phases inour dataset in DD to the reported phases in previously publishedstudies of mouse liver in LD (12, 15, 16, 18), we observed a correlation with a 1 to 2 h delay in DD (SI Appendix, Fig. S10 A and
C). The transcripts with the largest differences in delay in themRNA-­to-­peak-­protein abundance between LD and DD conditions included Nfil3, Per2, Per3, and Bmal1 (SI Appendix,
Fig. S10B). The differences resulted from a slightly earlier peak in
RNA abundance and ribosome binding in LD rather than a delayin the protein abundance peak; there was little difference between
LD and DD in the delay between RNA abundance and ribosome
binding (SI Appendix, Fig. S10D). Although these small differences
may reflect a true biological effect from light-­dependent changes
in transcription, they could also simply result from differences in
the algorithms used for phase determination, differences in experimental conditions, or both. Besides these 16 core circadian transcripts, most other transcripts with rhythmic ribosome binding inour dataset had a phase of around CT0 (SI Appendix, Fig. S6).
uORFs Suppress Translation. We also noticed a relationshipbetween ribosome occupancy and the presence of uORFs,
similar to previous reports (15, 18). Roughly half of all mousetranscripts contained at least one uORF (SI Appendix, Fig. S4B),
and we found rhythmic ribosome binding in 602 uORFs with an
adjusted P < 0.05 using JTK_cycle (Dataset S2). In particular, forcircadian transcripts, such as Cry1 and Bmal1, there appeared to
be increased ribosome binding in uORF regions, as measured byincreased ribosome profiling reads (Fig. 2A), although ribosome
binding in the 5′UTR was not always associated with uORFs
in circadian transcripts (SI Appendix, Fig. S4A). mRNAs withincreased numbers of uORFs had lower levels of ribosome
occupancy in the downstream CDS (Fig. 2B), whereas the lengthof the uORF and the distance of the uORF to the start codon did
not have a significant impact on ribosome binding in the CDS(SI Appendix, Fig. S4 C and D). To investigate whether uORFs
were sufficient to suppress translation in a combinatorial manner,
we created a luciferase reporter vector with multiple synthetic
uORFs. Predictably, increasing the number of uORFs reduced
luminescence from the reporter (Fig. 2C). Using this synthetic
reporter, we varied the position of a single uORF relative to the startcodon or the uORF length and found a higher degree of uORF-­
mediated repression the closer the uORF was to the start codon;
however, uORF length had no effect on repression (SI Appendix,
Fig. S4 E and F). Both the position of the uORF and the numberof uORFs altered the amplitude and mesor (mean luminescence)
without altering the period using two different promoters in cell-­based circadian luminescence assays (SI Appendix, Fig. S15). Other
factors, such as the Kozak consensus of the uORF, overlap with the
CDS, or uORF conservation may also impact repression strength(28, 29).
The Per2 uORF Suppresses Reporter Expression without Altering
Period. To further explore the impact of uORFs on circadianrhythms, we focused on the uORF in the circadian transcript

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Protein molecules per cell (thousands)

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Ribosome Profiling (RPKM)

Fig. 1. Measuring translation and protein from the same liver samples for 16 circadian mRNAs. (A) Time course of the quantified proteins and the correspondingtranslation level. Protein concentration (gray) is expressed as copies per cell assuming that the total amount of protein per cell is 0.5 ng for the average forall quantified peptides (10). Ribosome binding (red) is expressed as reads per kilobase of transcript per million mapped reads (RPKM). Data are mean ± SEM.
(B) Phase delay between ribosome binding and protein abundance. Copies per cell were averaged from all peptides for each protein. JTK analysis was used toestimate the phases for these averaged protein levels and for ribosome binding RPKM levels. (C) Linear regression analysis comparing ribosome binding toprotein amount averaged over 24 h showed that there was no correlation between ribosome binding and protein abundance (D and E) Linear regression analysis
comparing ribosome binding to protein amount at each sampled time point showed that some proteins like BHLHE40 (D) had high protein turnover or reduced
protein biosynthesis, whereas other proteins like PER2 (E) had low protein turnover or increased protein biosynthesis.

Per2. This uORF is evolutionarily conserved and consists of only
a start and stop codon (Fig. 3A and SI Appendix, Fig. S13), which
eliminates any potential effect of a translated uORF peptide on theregulation of Per2. Ribosomes bound the Per2 uORF rhythmicallyand slightly before peak ribosome binding on the Per2 transcript(Fig. 3B). Mutation of the uORF in Per2 increased the amplitude
of expression without affecting the phase or period in 3T3 cells
transfected with a luminescence reporter (Fig. 3 C and D). This
increase in amplitude was not affected by the amount of transfected
plasmid, inclusion of the full-­length Per2 5′UTR, or addition ofPER2 protein (SI Appendix, Fig. S5). CRISPR/Cas9-­mediatedmutation of the Per2 uORF in PER::LUC MEFs increased overall
luciferase expression levels compared to that of wild-­type MEFs;
however, we were unable to generate circadian rhythms in thesecells to determine the impact of the Per2 uORF on other circadianparameters such as period and phase (SI Appendix, Fig. S12).
To understand uORF-­mediated repression in individual cells,
we created a GFP fluorescent reporter plasmid driven by the Per2
promoter with or without a mutation in the Per2 uORF. We
transfected 3T3 cells with these plasmids and an mCherry normalization plasmid and analyzed the cells by FACS (SI Appendix,
Fig. S11). Mutation of the uORF increased GFP brightness without affecting mCherry brightness in individual cells (Fig. 3E), andincreased the GFP-­to-­mCherry ratio of the population (Fig. 3F)
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regardless of whether the cells were first gated on mCherry expression (Fig. 3 E and F) or GFP expression (SI Appendix, Fig. S11 C
and D). Mutation of the uORF also increased the total numberof GFP-­expressing cells without affecting the total number ofmCherry-­expressing cells (Fig. 3G). Thus, the Per2 uORF canrepress reporter expression within individual cells to control circadian amplitude.
Mice with a Mutation in the Per2 uORF have Reduced Sleep.

We used CRISPR/Cas9 to generate a knock-­in mouse harboringa mutation in the uORF of Per2, which removed both the startand stop codon of the uORF without disrupting a nearbyE’-­box (SI Appendix, Fig. S7A). Wild-­type and mutant mice were
phenotyped over 13 d in LD and then for 12 d in DD using the
Snappy Sleep Stager (30, 31), which is a respiration-­based method
to characterize sleep/wake parameters and circadian rhythms.
Both male and female Per2 uORF mutant mice had significantly
reduced sleep per day (mean ± SEM: 717 ± 19 min and 642 ±7 min, respectively) compared to their wild-­type littermates (774
± 11 min and 678 ± 4 min, respectively, P < 0.001 by two-­way
ANOVA) and a corresponding increase in wake duration per day(Fig. 4 A and B and SI Appendix, Fig. S8A) in LD conditions. There
was a decrease in sleep episode duration and a significant increasein the transition probability from sleep to awake termed Psw in Per2
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gacaaaTTGTAAgacaaaTTGTAAgacaaaTTGTAAgacaaaTTGTAA
gacaaaATGTAAgacaaaTTGTAAgacaaaTTGTAAgacaaaTTGTAA
gacaaaATGTAAgacaaaATGTAAgacaaaTTGTAAgacaaaTTGTAA
gacaaaATGTAAgacaaaATGTAAgacaaaATGTAAgacaaaTTGTAA
gacaaaATGTAAgacaaaATGTAAgacaaaATGTAAgacaaaATGTAAPer2 short promoter

0-4 uORFs linker

CMV promoter

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Fig. 2. uORFs suppress protein expression. (A) The 5′UTRs of Cry1ClockPer2, and Bmal1 transcripts contain at least one uORF (shaded regions below arrows).
The distribution of raw counts of ribosome profiling reads summed across all time points (black bars) for the 5′UTR of each transcript is shown. (B) Global
distribution of uORFs compared to ribosome profiling translation level (RPKM) shows that mRNAs with more uORFs are translated less compared to mRNAs withfewer uORFs. (C) Introduction of a variable number of synthetic uORFs represses relative luminescence from a Per2 short promoter in a dose-­dependent manner.

uORF mutant mice (SI Appendix, Fig. S8B); however, there were
no differences in other sleep parameters such as amplitude, Pws (thetransition probability from awake to sleep), or wake episode duration(SI Appendix, Fig. S8C). Per2 uORF mutant mice exhibited reduced
sleep duration particularly during the light-­to-­dark and dark-­to-­light transitions in the early morning and early evening (Fig. 4 C
and D). There were also differences in sleep duration later in themorning (SI Appendix, Fig. S8D) but not at other times during theday (SI Appendix, Fig. S8E). Next, we observed mice in constantdarkness over 12 d and found no differences in activity rhythms
or period (Fig. 4E); furthermore, there were less pronounced sleep
differences in DD compared to LD (SI Appendix, Fig. S9). Cosinor
analysis of the activity rhythms in LD and DD conditions revealed
no differences in period and amplitude between wild-­type and Per2
uORF mutant mice (SI Appendix, Fig. S9).
To understand the molecular mechanisms behind these phenotypic differences, we performed ribosome profiling and total
RNA sequencing of the liver from wild-­type and Per2 uORF
mutant male mice killed at ZT02-­04 (32) (SI Appendix, Figs. S18
and S19 and Datasets S4 and S5). Because Per2 levels are near
their lowest at ZT02-­04, we hypothesized that this time point
would show the largest difference in derepression of Per2 by mutation of the Per2 uORF. There was an increase in ribosome binding
in Per2 uORF mutant mice (SI Appendix, Fig. S19C), particularlyin the 5′UTR and around the start codon (SI Appendix, Fig. S19
D and E). Per2 RNA levels were also higher in the mutant miceby total RNA sequencing (SI Appendix, Fig. S19 C and H and
Dataset S5) and confirmed by qPCR analysis (SI Appendix,
Fig. S19B), although no differences were observed in Per2 translation efficiency (TE), which is defined as the ratio of ribosome
profiling reads to bulk RNA-­seq reads (SI Appendix, Fig. S19G
and Dataset S4). Protein levels were similar in Per2 uORF mutant
mice compared to that in wild-­type mice (SI Appendix, Fig. S19
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A and I). Taken together, these results suggest that Per2 mRNA
expression and ribosome binding is suppressed by the Per2 uORF,
which has a moderate impact on sleep duration.
Discussion
Global proteomics studies of mice livers revealed large numbers ofdiurnally rhythmic proteins. It has been estimated that 20 to 50%of these proteins are dependent on posttranscriptional and posttranslational mechanisms (8, 9, 11, 12, 33). Previously, we used
targeted proteomics to detect the rhythmicity and concentration of
a select number of low-­abundance core circadian proteins in constant darkness conditions (10). Here, we used ribosome profilingand quantitative mass spectrometry to understand the relationshipbetween those circadian protein rhythms and mRNA translation.
We found a correlation between the timing of protein production, as measured by ribosome profiling, and peak protein abundance for most transcripts (Fig. 1). However, BMAL1 and CLOCK
proteins had a delay between peak ribosome binding and protein
levels (Fig. 1) caused by delayed protein turnover via posttranslational modification pathways (34), rather than a delay in ribosome
binding (SI Appendix, Fig. S3). The peak protein abundance of PER
and CRY is also slightly delayed relative to their peak mRNA abundance (10, 19, 20), and we found that this was also due to protein
turnover rather than to a delay in ribosome binding (SI Appendix,
Fig. S3), similar to previous ribosome profiling studies (15, 16, 18).
There was a larger delay between the peak abundance of Cry2 ribosome binding and CRY2 protein than that of Cry1, which may
simply be a result of the lower amplitude of CRY2 rhythms.
We lacked the RNA-­seq data needed to compute TE in our circadian samples, which limited our ability to determine how uORF
features in 5′UTRs, such as number, length, and distance to CDS,
alter translation efficiency. However, using the closest publicly
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Luminescence (counts/min)-221 Per2 short promoter +75 chr1

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Fig. 3. The Per2 uORF is evolutionarily conserved and suppresses PER2 expression. (A) Genomic position of the Per2 short promoter, which contains an E’box
and uORF. Evolutionary conservation scores according to UCSC genome browser among 22 mammalian species in the Per2 5′UTR (black bars) with the uORF
highlighted (red bars). (B) Ribosome binding (RPKM) on the Per2 uORF (red) compared to the Per2 transcript (5′UTR, CDS, and 3′UTR, black). (C) Representative
bioluminescent traces showing that mutation of the Per2 uORF (ATGTAA to TTGTGA) increases the expression level of a pGL3-­P(Per2)-­dLuc luminescent reporter.
Reporter containing uORF (black), reporter with a mutant uORF (red). The shaded region is SD. (D) Cosinor analysis showing the normalized amplitude (Left),
period (Middle), and phase (Right) for cells transfected with the Per2 uORF (black) or a mutant Per2 uORF (red). Data from each group comprise at least 22 tracesfrom eight different experiments (see SI Appendix, Fig. S5, for the plots of individual traces for each experiment). (E) Representative scatterplot of GFP versus
mCherry expression for 3T3 cells transfected with a GFP-­fluorescence reporter with a Per2 uORF (+uORF, black) or a mutant Per2 uORF (−uORF, red) and amCherry-­normalization plasmid. The analyzed cells were gated on mCherry+ expression. (F) Histogram of the GFP/mCherry ratio for cells in E. (G) The percentage
of GFP+ cells (Left) was significantly higher in the −uORF population compared to the +uORF population, whereas the percentage of mCherry+ cells (Right) was
unchanged (n = 3 independent experiments). See SI Appendix, Fig. S11, for FACS gating strategy and analysis of cells from the GFP+ gate.

available RNA-­seq data (4) to our ribosome profiling data as a proxy
for transcript abundance (Dataset S3), we observed a decrease in TE
in transcripts with one or more uORFs (SI Appendix, Fig. S16), but
no relationship between TE and uORF length or uORF distance to
CDS (SI Appendix, Fig. S17).
Most transcripts with rhythmic translation in our dataset had aphase around CT0, corresponding to what would be thedark-­to-­light transition (SI Appendix, Fig. S6). Less than 10% of
the circadian translatome had significant rhythms in mRNA abundance according to JTK cycle analysis (P < 0.05), and these transcripts were enriched for circadian and metabolic processes byPanther gene ontology (GO) analysis (SI Appendix, Fig. S6 B and
C). Transcripts with a nonsignificant mRNA rhythmicity by JTK
cycle analysis (P > 0.05) also were biased toward CT0, but there
was no clear GO biological process underlying these rhythms
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(SI Appendix, Fig. S6 D and E). The set of transcripts with phases
between CT22 and CT02 (SI Appendix, Fig. S6 F and G) included
Npas2, Bmal1, Cry1, Nfil3, and Clock. These transcripts are known
to be regulated by the nuclear receptor NR1D1, also known as
Rev-­erbα, which transcriptionally represses many genes involved inmetabolism in a tissue-­and circadian-­dependent manner (35). In
the liver, NR1D1 predominantly binds genomic locations at
ZT08-­ZT10 with little-­to-­no binding at ZT22 (36–38). Thus,
NR1D1 derepression may be driving some of transcriptional, andsubsequent translational, peak observed in our data between CT22
and CT02. In addition, cyclic changes in translation machineryand polyadenylation (39, 40), translation activity of BMAL1 mediated by the mTORC1/S6K1 (41), or mTORC1 regulation by PER2(42) may also drive rhythmic translation of nonrhythmic mRNAtranscripts.
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B

Mutant (-uORF)

30

Female

20

4

6

8

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4

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0

25

*

30

10

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800

20

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= 23.8 0.8
= 23.7
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= 23.5
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3
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Sleep time ZT0–4 h (min)Sleep time ZT12–16 h (min)Downloaded from https://www.pnas.org by UNIVERSITY OF TOKYO MEDICAL LIBRARY on November 9, 2023 from IP address 157.82.128.3.

D2

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Sleep duration per hour (min)MaleFemaleWild type (+uORF)MaFe l emaleAFig. 4. Disruption of the Per2 uORF reduces sleep in mice. (A) Sleep duration per hour over 13 d in 12-­h light/12-­h dark (LD) conditions for Per2 uORF mutant
and wild-­type male and female mice. Each row indicates data from one mouse. (B) Mean sleep (Top) and wake (Bottom) duration over 24 h, averaged over 13 d.
Red, Per2 uORF mutant mice. Black, wild type. (C) Sleep duration per hour over 24 h in LD, averaged over 13 d for Per2 uORF mutant (red) and wild-­type (black)male (Top) and female (Bottom) mice. Lines indicate mean sleep duration at each time of day for each strain. Shaded area, SD at each time point. (D) Mean sleep
duration per hour averaged over 13 d during the early morning (Top) and early evening (Bottom). Per2 uORF mutant (red) and wild-­type (black) dots are individual
mice; lines indicate mean. (E) Representative double-­plotted actograms of sleep duration per 6 min bins of wild-­type and Per2 uORF mutant mice in constantdarkness (DD) over 12 d (Left) and corresponding periodograms (Right).

We observed widespread binding of ribosomes to uORFs in the
5′UTRs of many core circadian transcripts and found an inverse
relationship between uORF number and luciferase expression intransiently transfected reporter cells and CRISPR/Cas9-­generated
MEF cells (Fig. 2 and SI Appendix, Figs. S4, S12, and S15).
Approximately 50% of mouse and human mRNAs containuORFs, which are associated with widespread translational repression (29). Thus, uORF-­mediated suppression of translation may
constitutively reduce the abundance of circadian proteins or provide a posttranscriptional foothold to adjust protein abundanceby altering the activities of ribosome reinitiation factors (43–45).
Moreover, mutagenesis of uORFs has been shown to alter rhythmicity in other circadian systems (46, 47). Not all ribosome binding in 5′UTRs was associated with uORFs, and we observed
extensive ribosome binding in areas without an apparent uORF
for Cry1, Nr1d1, Clock, Dbp, and Per3 (Fig. 2 and SI Appendix,
Fig. S4). For the Nr1d1 5′UTR, we observed close overlap in the
nucleotides necessary for internal ribosome entry site (IRES)-­
mediated translation (48) and ribosome binding (SI Appendix,
Fig. S14A) and confirmed IRES-­mediated translation from the
Nr1d1 5′UTR but not the Cry1 5′UTR (SI Appendix, Fig. S14B).
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Near-­cognate uORF translation (from a non-­AUG start codon)
may also drive ribosome binding (14). In silico mapping ofnear-­cognate start codons with ribosome binding data revealed
numerous potential near-­cognate uORFs (SI Appendix, Fig. S14C)
but experimental validation is still needed.
The uORF in the Per2 5′UTR is an attractive target to understand the role of uORFs in circadian biology, not only because
cis-­elements within the Per2 promoter are well understood (49–52)
but also because the Per2 uORF is too short to encode a peptide.
PER2 production is posttranscriptionally controlled by miRNAs(53, 54), antisense transcription (55), and hnRNP1-­mediatedmRNA degradation (56), which suggests that Per2 posttranscriptional control is important for producing the optimal amount of
PER2 protein. Mutation of the Per2 uORF increased the amplitudeof a reporter plasmid without affecting the phase or period (Fig. 3),
similar to effects observed by mutating the Per2 antisense transcript(57).
Both the abundance and timing of Per2 expression are criticalfor maintaining circadian rhythmicity in mice because constitutive expression of PER2, unlike CRY1, disrupts behavioral
rhythms (58). Several posttranscriptional mechanisms such aspnas.org

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Per2 antisense transcription (55) and miRNAs (53, 54, 59) in the
Per2 genomic locus alter the timing and abundance of PER2
protein expression. For example, replacing the Per2 3′UTR withan SV40 late poly(A) signal greatly amplifies bioluminescencerhythms in PER2:LUC mice and increases free-­running periods(54). Mutation of the Per2 uORF increased amplitude in reporter
cells (Fig. 3) but did not increase the free-­running period in mice
(Fig. 4). Per2 uORF mutant mice did have reduced total sleep in
LD conditions (Fig. 4), as observed in Per1/Per2 double mutant
mice under similar sleep phenotyping conditions (30), but nosignificant change in sleep in DD conditions (SI Appendix,
Fig. S9). Although our Per2 mutation abolished the canonical
ATGTGA uORF in the Per2 5′UTR, it also introduced anear-­cognate uORF (CTGTAG) two nucleotides upstream of theoriginal uORF. We think that this near-­cognate uORF was nonfunctional because we observed increased Per2 ribosome binding
and mRNA levels in Per2 uORF mutant mice compared to thatin wild-­type mice (SI Appendix, Fig. S19) and increased luciferase
expression in mutant PER2:LUC MEF cells (SI Appendix,
Fig. S12). One possible explanation for the rise in Per2 mRNAlevels is uORF-­triggered nonsense-­mediated decay (NMD) (60).
In this scenario, mutation of the Per2 uORF derepresses NMD
of Per2, which results in an increase in Per2 mRNA. However,
Per2 mRNA expression does not increase upon knockdown of
the NMD component Smg6 (61), which suggests that other proteins in NMD independent of SMG6 may be involved (62).
Another group deleted the start codon of the Per2 uORF but
found no effect on Per2 mRNA levels and did not perform ribosome profiling (63), so it is unclear whether ribosome binding in
Per2 is elevated in their Per2 uORF mutant mice as it is in ourmutant mice. In both studies, PER2 protein levels in the mutant
mice were similar to that in wild-­type mice, indicating that circadian proteostasis pathways compensate for the increase in PER2
protein production. In LD conditions, sleep in our Per2 uORF
mutant mice was particularly reduced during light-­to-­dark anddark-­to-­light transitions (Fig. 4), which may result from a lower
sleep episode duration and an increase transition probability fromsleep to wake (SI Appendix, Fig. S8B). Regulation of PER2 stability affects sleep (64, 65), and sleep deprivation reciprocallyaffects Per2 expression (66–69) providing a rationale for how
increased Per2 expression could disrupt sleep in our mice.
Mutation of the E’box cis-­element, which is only a few base pairs
upstream of the Per2 uORF, abolishes molecular oscillations inmutant cells without disrupting the free-­running period in mice(49). These mice re-­entrain quicker under an artificial jetlag experiment than wild-­type mice, and it will be interesting to observe
how Per2 uORF mutant mice behave under similar conditions.
Further studies, particularly at a neurological level, are also needed
to understand the precise mechanism by which sleep is reducedin these animals.
Materials and Methods
Animals. All animal experiments were approved by the Institutional Animal Careand Use Committee of the RIKEN Kobe branch. Eight-­to-­ten-­week-­old wild-­type
male mice (C57BL/6N, Japan SLC) were entrained under 12-­h light (400 lx) 12-­hdark (LD) for 2 wk. Twenty-­four hours after transferring to constant darkness (DD),wild-­type mice were killed every 4 h over one day (CT0, CT4, CT8, CT12, CT16,
CT20, and CT24) for ribosome profiling analysis as in ref. 10. Livers were excised,snap-­frozen in liquid nitrogen, and stored at −80 °C until use. For details regarding Per2 uORF mutant mouse construction, see SI Appendix.
Plasmids. For details regarding plasmid construction, see SI Appendix.
Ribosome profiling. Ribosome profiling was performed essentially as describedin ref. 24. Frozen liver samples (~50 mg) were pulverized and then homogenizedin 400 µL polysome lysis buffer (150 mM NaCl, 20 mM Tris–HCl pH 7.4, 5 mM
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MgCl2, 5 mM DTT, 100 µg/mL cycloheximide, 1% Triton X-­100, and 25 U/mL Turbo
DNAse I). Lysates were incubated on ice for 5 to 10 min, triturated through a 26-­G
needle 10 times, and clarified by centrifugation (20,000 × g for 10 min at 4 °C).
About 300 µL of the supernatant was transferred to a new tube. Unbound RNA
was digested by the addition of 7.5 µL RNAse I (ThermoFisher) for 45 min andthen stopped by 10 µL SUPERase In RNAse Inhibitor (ThermoFisher). The digestion was transferred to 13 mm × 51 mm polycarbonate ultracentrifuge tubes,
layered on top of 0.9 mL sucrose cushion (150 mM NaCl, 20 mM Tris–HCl pH 7.4,
5 mM MgCl2, 5 mM DTT, 100 µg/mL cycloheximide, 1 M sucrose, and 10 U/mL
SUPERase In), and centrifuged in a TLA100.3 rotor at 70,000 rpm at 4 °C for 4 h.
Ribosome pellets were resuspended in 0.7 mL Qiazol, and mRNA was recovered
using the miRNeasy RNA extraction kit (Qiagen) according to the manufacturer’s
instructions. For library construction and sequencing, see SI Appendix.
Bioinformatic analysis of ribosome profiling. For details regarding sequence
processing and alignment, see SI Appendix. For each transcript, nonoverlapping
five-­codon windows were tiled across the coding region, and the transcript wasconsidered well-­translated if it had a median value of at least two reads per window (excluding the excluding the first fifteen and the last five codons). Using all
well-­translated transcripts, we created file where each transcript is represented
across all samples by its RPKM value. We used this file in JTK_CYCLE (26) to
identify all rhythmic transcripts. We used a threshold on the adjusted P < 0.05
to assess significance.
To detect uORFs, we processed the 5′UTR of all transcripts, extracted theirsequence, and identified all pairs of a start codon (AUG) and a stop codon (UGA,
UAA, or UAG) in phase with each other. Using the same read assignment method
as in SI Appendix, we also allocated reads aligned to the UTR to their corresponding uORF (where appropriate).
Cells. NIH3T3 and MEFs from PER2::LUCIFERASE (PER2::LUC) knock-­in reporter
mice (70) were cultured in DMEM (ThermoFisher), 10% FBS (JRH Biosciences),and 1% penicillin/streptomycin (ThermoFisher) at 37 °C in 5% CO2. For additional
details, see SI Appendix.
Real-­time circadian luciferase assay. Real-­time circadian luciferase assays
were performed as previously described (71). Briefly, the day before transfection,
NIH3T3 cells or PER2:LUC MEFs were plated onto 35-­mm dishes at a density 4 ×
105 per well. The following day, NIH3T3 cells were cotransfected using FuGene6(Roche) with 1 µg of the indicated luciferase reporter plasmid according to the
manufacturer’s instructions (n = 3) and cultured at 37 °C. After 72 h, the media
in the well were replaced with 2 mL of DMEM containing 10% FBS supplemented
with 10 mM HEPES (pH 7.2, ThermoFisher), 0.1 mM luciferin (Promega), antibiotics, and 10 µM forskolin (Fermentek, NIH3T3) or 100 µM dexamethasone(ThermoFisher, PER2::LUC MEFs). Luminescence was measured by a photomultiplier tube (LM2400R, Hamamatsu Photonics) for 1 min at 12 min intervals ina darkroom at 30 °C.
Dual-­luciferase assay. NIH3T3 cells were plated on 6-­well plates at a densityof 2 × 105 per well. The following day cells were cotransfected with 0.95 µg of aFirefly luciferase reporter plasmid and 50 ng phRL-­SV40 plasmid (Renilla luciferase, Promega) as an internal control for transfection efficiency using FuGene6(Roche). Cells were harvested and assayed by the Dual-­Luciferase Reporter
Assay System (Promega) according to the manufacturer’s instructions 48 h after
transfection.
Preparation of mouse nuclear lysate and immunoblot analysis. Mice were
killed by cervical dislocation, and livers were dissected at CT2-­4, snap-­frozen inliquid nitrogen, and stored at −80 °C. Liver extracts were prepared according toref. 72 with minor modifications. See SI Appendix for further details.
FACS. NIH3T3 cells were plated onto plastic 35-­mm dishes or 35-­mm imaging
dishes (Ibidi) at a density 4 × 105 per well. The following day, cells were cotransfected using FuGene6 (Roche) with 0.5 µg of the indicated fluorescence reporter
plasmids (1 µg total) according to the manufacturer’s instructions (n = 3) andcultured at 37 °C. After 72 h, cells were trypsinized, sorted using FACSAria I (BD
Biosciences), and analyzed by FlowJo version 10.8.1.
Sleep phenotyping. Sleep phenotyping was conducted in 12-­week-­old (LD) and14-­week-­old (DD) mice in a Snappy Sleep Stager (SSS) using Per2 uORF mutant
mice and wild-­type littermates as a control (30). SSS is a noninvasive, respiration-­based sleeping staging system in which mice are placed in a chamber connected
to a respiration sensor that detects pressure differences between the outside andinside of the chamber. Detailed methods have been described previously (30).
For details regarding sleep and wake parameters, see SI Appendix.
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Statistical analyses. Statistical analyses were performed in R version 3.4.3,
Prism 7.0, and custom Jupyter notebooks version 6.3. Two-­way ANOVA and aStudent’s t test were used to test differences in sleep parameters between wild-­
type and Per2 uORF mutant mice. For data analyzed by the Student’s t test, thedata were first confirmed to have a Gaussian distribution by the Shapiro–Wilk
normality test, and equal variance within this distribution was confirmed by an F
test. Lomb–Scargle periodograms were implemented using SciPy version 1.7, andcosinor analysis was implemented using CosinorPy (73). Statistical significancewas defined as *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. for not significant.
Data, Materials, and Software Availability. NGS data have been deposited in

GEO (GSE201732 (25), GSE231820 (32)). Previously published data were used
for this work [Data from mass spectrometry (Fig. 1A) and qPCR (SI Appendix,
Fig. S3A) was previously published by our lab in Narumi et al., 2016 in PNAS.].
We thank Ryohei Narumi for the circadian liver
samples and for the use of the mass spectrometry and qPCR data and
Dr. Shigehiro Kuraku and the Genome Information Research Center at Osaka
University for sequencing of the ribosome profiling libraries. We thankDr. Shihoko Kojima and Dr. Seung-­Hee Yoo for PER2::LUC MEFs. We thank all lab
members at the RIKEN Center for Biodynamics Research, in particular, Natsumi
Hori and Yumika Sugihara for the production of mutant mice, and Masako Kunimi
and Ruriko Inoue for breeding and helping with the phenotype measurements ofthe mutant mice. We thank all members of the Systems Immunology Laboratory
and the Laboratory for Host Defense at the Immunology Frontier Research Center,in particular, Kitiya Piboonprai for help with genotyping and Shizuo Akira for hiskind support. This work was also supported by grants from Exploratory Research

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https://doi.org/10.1073/pnas.2214636120

for Advanced Technology grant (JPMJER2001, H.R.U.) from the Japan Scienceand Technology Agency, KAKENHI Grant-­in-­Aid from the Japan Society for the
Promotion of Science (Scientific Research S 18H05270 to H.R.U., Scientific Research
C JP21K06385 to R.G.Y.), Human Frontier Science Program Research Grant Program(HFSP RGP0019/2018, H.R.U.), Ministry of Education, Culture, Sports, Science andTechnology Quantum Leap Flagship Program (JPMXS0120330644), Japan Agency
for Medical Research and Development (JP20am0401011), and the intramural
Grant-­in-­Aid from the RIKEN Center for Biosystems Dynamics Research to H.R.U.
This research was supported by the Research Support Project for Life Science and Drug
Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research)from the Japan Agency for Medical Research and Development under Grant Number
JP22ama121025. This work was supported by a Human Frontiers Science Program
postdoctoral fellowship to A.M. and a Japan Society for the Promotion of Science
Grant-­in-­Aid for Early Career Scientists (18K14755 to A.M.). Two children were born
to A.M. during the course of this project, and he thanks them and Mari Nishino for
their care, love, and support.

Author affiliations: aLaboratory for Synthetic Biology, RIKEN Quantitative Biology Center,
Suita, Osaka 565-­0871, Japan; bLaboratory for Host Defense, Immunology Frontier
Research Center, Suita, Osaka 565-­0871, Japan; cLaboratory for Systems Immunology,
Immunology Frontier Research Center, Suita, Osaka 565-­0871, Japan; dLaboratory of
Animal Genetics and Breeding, Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya 464-­8601, Japan; eSchool of Computer Science, Queensland Universityof Technology, Brisbane QLD 4000, Australia; fCentre for Data Science, Queensland
University of Technology, Brisbane QLD 4000, Australia; and gDepartment of Systems
Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-­0033,
Japan

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Supporting Information for
Circadian ribosome profiling reveals a role for the Period2 upstream
open reading frame in sleep.
Arthur Millius, Rikuhiro Yamada, Hiroshi Fujishima, Kazuhiko Maeda, Daron M. Standley,
Kenta Sumiyama, Dimitri Perrin, and Hiroki R. Ueda
Hiroki R. Ueda
Email: uedah-tky@umin.ac.jp

This PDF file includes:
Supporting text
Figures S1 to S21
Tables S1 to S3
Legends for Datasets S1 to S5
SI References
Other supporting materials for this manuscript include the following:
Datasets S1 to S5

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Supporting Information Text

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Methods
Animals. Per2 uORF mutant (uORF: ATGTGA to mutant uORF: GTAGGT) mice weregenerated by one-cell embryo microinjection of synthesized Cas9 mRNA, gRNA (5'TTTCCACTATGTGACAGCGGAGG-3'), and ssODN (5'GTCACGTTTTCCACTGTAGGTCAGCGGAGGGCGACG-3') in C57BL/6N fertilized
eggs, and genotypes were confirmed by Sanger sequencing.
Plasmids. The pGL3-P(Per2)-dLuc reporter plasmid (1) was modified by standard PCRmutagenesis to generate pGL3-P(Per2)-uORF-dLuc (ATG->TTG mutation in the Per2
uORF). Synthetic oligonucleotide linkers containing 0-4 artificial uORFs (Fastmac, Japan)
were inserted into a PCR-linearized pGL3-P(Per2)-dLuc and pGL3-P(SV40)-3xE'box-dLuc(2) by In-Fusion cloning (Takara Bio). Plasmids containing full-length Per2 5'UTR andPER2 were cloned from mouse cDNA (Clone PX00938 H17, RIKEN) and pMU2-PER2-Luc(3), respectively, and inserted into pGL3-P(Per2)-dLuc or pGL3-P(Per2)-uORF-dLuc by InFusion cloning to create pGL3-P(Per2)-5'UTR-dLuc and pGL3-P(Per2)-5'UTR-PER2-dLuc
and the respective uORF mutant plasmids. Oligos for Per2 sgRNA (5'caccgTTCCACTATGTGACAGCGGA and 5'-aaacTCCGCTGTCACATAGTGGAAc) wereannealed and inserted into BbsI-digested pSpCas9(BB)-2A-Puro (Addgene #62988) or
pSpCas9n(BB)-2A-Puro (Addgene #62987) to make pCas9-Per2 or pCas9n-Per2,
respectively. The promoter sequences from pGL3-P(Per2)-dLuc or pGL3-P(Per2)-uORFdLuc were cloned into pD1EGFP-N1 (Takara Bio) to create pD1-P(Per2)-EGFP or pD1P(Per2)-uORF-EGFP, respectively. mCherry was PCR-amplified and blunt-end ligated into
inverse-PCR amplified pD1-P(Per2)-EGFP for the mCherry version. The Nr1d1 5'UTR(nucleotides 1-629 in NM_145434.4) and Cry1 5'UTR (nucleotides 1-583 in NM_007771.3)
were inserted into phpRL-BCL2-FL-pA (Addgene #42595) by In-Fusion cloning to create
phpRL-Nr1d1 5'UTR-pA and phpRL-Cry1 5'UTR-pA, respectively. All plasmid sequenceswere verified by Sanger sequencing.
Ribosome Profiling. RNA was precipitated from the elution by the addition of 38.5 µl water,
1.5 µl GlycoBlue (ThermoFisher), and 10 µl 3 M sodium acetate pH 5.5 followed by 150 µl
isopropanol, stored overnight at -80 °C, pelleted by centrifugation (20,000 × g for 30 min at
4 °C), and resuspended in 5 µl 10 mM Tris pH 8. Approximately 2–4 µg RNA was separated
on a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 65 min in 1× TBE, andfragments between 26 and 34 nt were excised and recovered in 400 µl RNA gel extraction
buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA, and 0.25% (wt/vol) SDS) overnight.
RNA was precipitated by the addition of 1.5 µl GlycoBlue and 500 µl, stored at -80 °C for>30 min, pelleted by centrifugation (20,000 × g for 30 min at 4 °C), and resuspended in 10 µl
10 mM Tris pH 8. RNA was dephosphorylated by T4 PNK (Takara Bio), precipitated andrecovered as previously described, resuspended in 8.5 µl 10 mM Tris, and 1.5 µl of
preadenylated and 3' blocked miRNA cloning linker(1/5rApp/CTGTAGGCACCATCAAT/3ddC/, IDT) was added. The linker mixture wasdenatured for 90 s at 80 °C, cooled to room temperature, ligated by T4 Rnl2 (NEB) for ~5 hat room temperature, precipitated, recovered, and separated by 15% TBE-urea gelelectrophoresis. Ligation products were recovered overnight in RNA gel extraction buffer,precipitated, and recovered in 10 µl 10 mM Tris pH 8. Ligated RNA was reverse transcribedat 48 °C for 30 min using SuperScript III (ThermoFisher) with the reverse transcription
primer (5′-(Phos)AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC2

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(SpC18)-CACTCA(SpC18)- TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3′, where
SpC18 indicates a hexa-ethyleneglycol spacer). Reverse transcription products wereprecipitated, recovered, separated by 15% TBE-urea gel electrophoresis, and recovered
overnight in DNA gel extraction buffer (300 mM NaCl, 10 mM Tris pH 8, and 1 mM
EDTA). The cDNA was precipitated, recovered, resuspended in 15 µl 10 mM Tris pH 8, andcircularized by CircLigase (EpiCentre) for 1 h at 60 °C. rRNA was depleted by combining 5µl circularization reaction with 1 µl of subtraction oligo pool, 1 µl 20× SSC, and 3 µl water,incubating at 37 °C for 15 min and binding to MyOne Streptavidin C1 DynaBeads(ThermoFisher) at 37 °C for 15 min with mixing at 1000 rpm. Eluate was precipitated,recovered, and resuspended in 5 µl 10 mM Tris pH 8. rRNA-depleted cDNA was PCRamplified by Phusion High-Fidelity DNA Polymerase (NEB) in a 20 µl reaction (denature
94 °C for 15 s, anneal 55 °C for 10 s, extend 72 °C for 10 s) for 12-16 cycles. PCR products
were separated on an 8% TBE gel, recovered overnight in DNA gel extraction buffer,precipitated, recovered, and resuspended in 15 µl 10 mM Tris pH 8. Libraries were quantified
using the high-sensitivity DNA chip on the Agilent BioAnalyzer according to the
manufacturer's protocol, pooled, and amplified on the Illumina HiSeq or NovaSeq6000
system according to the manufacturer's protocol.
Bioinformatic analysis of ribosome profiling. After quality control using FASTQC(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), reads were clipped using
fastx_clipper (with parameters -Q33 -a CTGTAGGCACCATCAAT -l 25 -c -n) andprocessed with fastx_trimmer (with parameters -Q33 -f 2), both from the FASTX-Toolkit(http://hannonlab.cshl.edu/fastx_toolkit/). We then used Bowtie 2.1.0.0 (4) to map theresulting reads to rRNA sequences. Reads that were successfully aligned to these sequences
are discarded, and only unaligned reads were used for downstream analysis. These reads that
did not align to rRNA were mapped to the mouse mm10 reference genome. Using samtools
0.1.19.0 and egrep, we extracted reads for which an exact match between the sequence andthe reference could be found.
Using reads aligned to a unique location, we then calculated transcript density profiles. Thisrelies on assignment each footprint alignment (i.e., each read) to a specific A site nucleotide
based on the length of the fragment. The initial assumption is that this site will be close to thecenter of the read. To calculate the best offset, we considered a metagene that captured allreads and their position relative to the start site and calculated the offset that optimized
phasing. The best results were obtained when the offset from the 5' end of the alignment was:
26 nt long, +13; 27-28 nt long, +15; 29-30 nt long, +16; 31-32 nt long, +17. Reads shorter
than 26 or longer than 32 nucleotides were discarded. Using this offset, we assigned eachread to a unique nucleotide, and constructed transcript density profiles by counting thenumber of reads whose A site was assigned to each nucleotide position. Raw data has been
deposited in GEO (GSE201732 and GSE231820).
Cells. PER2::LUC MEFs were plated in 10 cm dishes at a density 4 × 105 per well,transfected the next day with pCas9-Per2 or pCas9n-Per2 and 560 bp of single-stranded
linearized DNA (Guide-it Long ssDNA Production System, Takara Bio) containing
mutations in the Per2 uORF (ATGTGA to GTAGGT), and after 48-h selected in 1 µg/ml
puromycin for puromycin-resistant clones according to (5). Individual clones were expanded,sequence-verified, and clones containing at least one allele with a mutation in the Per2 uORF
were used for downstream analysis.

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Preparation of wild-type and Per2 uORF mutant mouse lysate for ribosome profiling,
total RNA sequencing, qPCR, and immunoblot analysis. Approximately 0.1 g mouse livertissue from wild-type and Per2 uORF mutant male mice sacrificed at ZT02-04 was washedrepeatedly in ice-cold PBS (Nacalai), resuspended in 1 ml polysome lysis buffer (150 mM
NaCl, 20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 5 mM DTT, 100 µg/ml cycloheximide, 1%
Triton X-100, 25 U/ml Turbo DNAse I), and homogenized by glass Dounce
microhomogenizer (>10 strokes tip A, then >10 strokes tip B). The homogenate was clarified
by centrifugation (20,000 × g for 10 min at 4 °C) and the supernatant divided – 300 µl wasimmediately used for ribosome profiling library preparation as above, 200 µl was added to 1
mL TRIzol (Nacalai) for total RNA sequencing, and the remaining was snap frozen andstored at -80 °C for subsequent western blot and qPCR analysis. Total RNA was extracted
and analyzed using the Agilent 2100 bioanalyzer according to the manufacturer’s
instructions. Total RNA libraries were prepared using the TruSeq stranded mRNA kit(Illumina) according to the manufacturer’s instructions.
Preparation of nuclear and cytoplasmic extracts. Extracts were prepared essentially as
described previously (6) with some modifications. Approximately 0.1 g mouse liver tissue
from wild-type and Per2 uORF mutant male mice sacrificed at ZT02-04 was washedrepeatedly in ice-cold PBS (Nacalai), resuspended in 1 ml per 0.1 g tissue in homogenizationbuffer (5 mM KCl, 10 mM Tris-HCl pH 7.4, 2 mM EDTA, 1 mM DTT, 1× cOmplete Mini
EDTA-free protease inhibitor tablet [Sigma]) + 2.2 M sucrose, and homogenized by glass
Dounce microhomogenizer (>10 strokes tip A, then >10 strokes tip B). Approximately 550 µllysate was layered on 1.5 ml sucrose cushion (homogenization buffer + 2.2 M sucrose + 10%
glycerol) in 13 mm × 51 mm polycarbonate ultracentrifuge tubes, and centrifuged in a
TLA100.3 rotor at 70,000 rpm at 4 °C for 45 min. The supernatant was removed and used as
the cytoplasmic extract. The nuclear pellet was rinsed once glycerol without disturbing thepellet with homogenization buffer + 10% glycerol, then resuspended in 150 µl
homogenization buffer + 10% glycerol, transferred to a new tube containing 150 µl urea
buffer (2 M urea, 600 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, 1× cOmplete Mini EDTAfree protease inhibitor tablet [Sigma]), incubated on ice for 20 min with occasional mixing,
and then centrifuged (20,000 × g for 10 min at 4 °C). This supernatant was used as thenuclear extract.
Western blot. Total protein lysates were thawed and diluted to ~10 mg/ml in polysome lysis
buffer. Cytoplasmic and nuclear lysates were diluted to 1 mg/ml and 0.6 mg/ml, respectively,
in homogenization buffer + 10% glycerol. Lysates were separated by SDS-PAGE, transferredto nitrocellulose membrane (Thermofisher) by semidry transfer (Bio-Rad), blocked in 5%(wt/vol) skim milk in TBST (50 mM Tris pH 7.5, 140 mM NaCl, 0.1% Tween) for 1 h at
22 °C, and then incubated overnight at 4 °C in anti-PER2 antibody (1:1000, PM083, Medical& Biological Laboratories) or anti-histone H3 antibody (1:1000, 9715, Cell Signaling). Afterwashing in TBST, blots were incubated in anti-rabbit IgG HRP-conjugated secondary
antibody (1:2000, NA934, Cytivia) or mouse beta-actin conjugated-HRP antibody (0.8:2000,
sc-47778, Santa Cruz Biotechnology) for 1 h at 22 °C and developed by Immobilon Forte
Western HRP (Millipore).
qPCR. Approximately 100 µl lysate was extracted in 1 ml TRIzol (Nacalai) according to the
manufacturer’s instructions and re-suspended in 50 µl 10 mM Tris pH 8.0. RNA was digested
with TURBO DNAseI (Thermofisher) for 1 h at 37 °C and recovered with an equal volume
1:1 phenol:chloroform containing isoamyl alcohol pH 5.2 (Nacalai). After centrifugation(20,000 × g for 5 min at 4 °C), approximately 37.5 µl of the aqueous layer was precipitated
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with 10 µl 3 M sodium acetate pH 5.5 and 1.5 µl GlycoBlue followed by 150 µl isopropanol.
Samples were frozen at -80 °C for >30 min, centrifuged (20,000 × g for 30 min at 4 °C),
washed in 500 µl 75% ethanol, dried for 10 min at 37 °C, and RNA was resuspended in 15 µl
10 mM Tris pH 8.0. Approximately 500 ng RNA was reverse transcribed using ReverTra
Ace qPCR RT Master Mix (Toyobo) and amplified using Thunderbird SYBR qPCR mix(Toyobo) with Per2 (forward: 5'-GCACATCTGGCACATCTCGG-3'; reverse: 5'TGGCATCACTGTTCTGAGTGTC-3') and actin (forward: 5'CACTGTCGAGTCGCGTCCA-3'; reverse: 5'-CATCCATGGCGAACTGGTGG-3') primers
on ViiA 7 (Applied Biosystems). Relative Per2 mRNA was quantified using the CT
method with actin as a reference gene.
Sleep and wake parameters. Sleep and wake duration are the mean total sleep and wake
duration, respectively, per day over 13 d. Psw is the transition probability from sleep toawake; Pws is the transition probability from awake to sleep. Mathematically, Ei is the i-th
epoch, which is either sleep (s) or wake (w). Let NXY (X,Y ∈{w,s}) be the number of elementsof the set {(Ei, Ei+1) | Ei = X, Ei+1 = Y}. Pws and Psw are defined as Nws/(Nws + Nww) andNsw/(Nsw + Nss), respectively. The amplitude is defined as the coefficient of variation of sleep
time for each 10-min bin over 24 h.

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Supplementary Figure 1 | Analyzing mRNA expression, translation activity, and3absolute protein copy number levels in the same mice liver samples over the course of a4day (related to Figure 1). (A) All mice were carefully kept and handled according to the5

RIKEN Regulations for Animal Experiments. Eight to ten-week-old-male mice (C57BL/6N,

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Japan SLC) were entrained under a 12–12 h light-dark (LD) conditions (400 lux) for 2 weeks.

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After transferring to constant darkness (DD), mice were sacrificed every 4 h over 1 day (i.e.

8CT0, 4, 8, 12, 16, and 20) for proteomic analyses, every 4 h over 1 day (i.e. CT0, 4, 8, 12, 16,

920, and 24) for ribosome profiling analyses, or every 4 h over 2 days (i.e. CT0, 4, 8, 12, 16,

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20, 24, 28, 32, 36, 40, and 44) for qPCR analyses. (B) Two mice livers at seven different

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circadian time points were lysed and ribosomes were isolated by a sucrose cushion.

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Ribosome-protected mRNA fragments were extracted, reverse transcribed, and circularized

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into a library. Libraries were depleted of the common most abundant rRNA, PCR amplified

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and sequenced (~1.02 billion reads). (C) Schematic description of the MS-QBiC workflow. A

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purification-tag, a quantification-tag, and a tryptic peptide of the target protein (target

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peptide) were sequentially arrayed as a single peptide sequence (MS-QBiC peptide). The

17target peptide sequence was attached by one- or two-step PCR. The MS-QBiC peptide was

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synthesized in the PURE system in the presence of stable isotope-labeled Arg and Lys for

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isotopic labeling both the quantification-tag and the target peptide. Trypsin digestion of20

purified MS-QBiC peptide produced equal amounts of isotopically labeled quantification-tag

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and target peptide. The quantification-tag was used to measure purified MS-QBiC peptide2

and the target peptide was used as an internal standard for target protein quantification.

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Supplementary Figure 2 | Validation of ribosome profiling cDNA libraries (related to3

Figure 1). (A) Read distribution within 5'UTRs, CDS, and 3'UTRs compared with the size of

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those features. Ribosome profiling reads are enriched for the CDS and 5'UTR, and relatively

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few reads map to the 3'UTR. (B) Frame analysis of ribosome profiling reads of single protein

6isoform mRNAs shows a preference for frame 1 (the coding frame). Box-and-whisker plots:

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midline, median; box, 25th and 75th percentiles. Whiskers are minimum and maximum

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values. (C) Box and whisker plots show the distribution of read lengths from all sequences.

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The majority of reads were between 27 and 32 nt, which matches the footprint size of the

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ribosome. (D) Summary of mapped reads. (E) Correlation of RPKM between replicate 1 and11

replicate 2 at CT0 shows a high degree of correlation in ribosome binding between samples.

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Supplementary Figure 3 | Measuring translation and RNA expression from the same3

liver samples for 16 circadian mRNAs (related to Figure 1). (A) Time course of the4quantified RNA expression levels (grey) by qPCR derived from data in (7) and the5corresponding ribosome binding (RPKM, red). Error bars are the standard deviation of the6

two replicates. (B) Phase delay between mRNA expression and ribosome binding. Absolute

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cDNA abundance was calculated using the standard curve obtained from mouse genomic

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DNAs, and JTK analysis was used to estimate the phase of each mRNA.

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Supplementary Figure 4 | Distribution of upstream open reading frames in clock3

transcripts (related to Figure 2). (A) The 5' UTRs of Bhlhe40, Ck1e, Ck1d, Dbp, Tef, Nfil3,

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Nr1d1, Nr1d2, Per1, Cry2, Per3, and Rorc transcripts, location of uORFs (shaded regions),10

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and raw read counts from the ribosome profiling data (black bars). (B) The number of2transcripts with a given number of uORFs. (C) The relationship between the length of an

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uORF and ribosome binding (RPKM) in the corresponding ORF. (D) The relationship4

between the distance of an uORF to the ORF start codon and ribosome binding (RPKM) in5

the corresponding ORF. (E) Introduction of a single uORF in the Per2 short promoter of6

different lengths does not alter the relative luminescence of the Per2 luciferase reporter. (F)7The distance of a single uORF in the Per2 short promoter to the start codon correlates with8

the relative luminescence of the Per2 luciferase reporter.

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Supplementary Figure 5 | Detailed analysis of the Per2 uORF (related to Figure 3). (A)3

Bioluminescent recording of 3T3 cells transfected with the Per2 short promoter containing a

4wild-type (black) or mutant (red) Per2 uORF at 1 µg (A) and 0.4 µg (B) transfected plasmid.

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Each graph is an independent experiment performed on a different day; multiple traces on the6same graph indicate different transfected plates. Data from the upper middle panel of (B) was
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1used for Fig. 3B. (C) Cosinor analysis showing normalized mesor for the bioluminescent2

traces in (A) and (B). (D) Mutation of the Per2 uORF (ATGTAA to TTGTGA) with the full-3

length Per2 5' UTR (red) increases the luminescence expression level of the reporter as

4measured by bioluminescent recording compared to that of a reporter containing the uORF

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(black). Shaded region is SD. (E) Mutation of the Per2 uORF (ATGTAA to TTGTGA) with6

the full-length Per2 5' UTR and PER2 protein (red) increases the luminescence expression

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level of the reporter as measured by bioluminescent recording compared to that of a reporter

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containing the uORF (black). Shaded region is SD. (F) Relative luminescence experiments of

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3T3 cells transfected with (+uORF) or without (-uORF) the uORF in pGL3-P(Per2)-dLuc.

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Supplementary Figure 6 | Phase analysis (related to Figure 3). (A) Heat map of ribosome

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profiling rhythmic transcripts identified by JTK cycling analysis (P < 0.05) showing RPKM

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data over 24 hours for 2952 genes. Transcripts are sorted by phase, and RPKM values are5normalized within each gene (row). Note that most transcripts peak at CT0 (red bars in first6

two columns). (B) Polar histogram showing the ribosome profiling (left) and RNA (right)
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1phase distribution of the 265 ribosome profiling rhythmic and mRNA rhythmic transcripts.

2(C) Panther analysis of gene ontology biological processes based on genes in (B) showing an

3increase in circadian rhythm regulation pathways and metabolic processes. (D) Polar

4histogram showing the ribosome profiling phase distribution (left) of the 2687 ribosome

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profiling rhythmic transcripts that did not reach significance in mRNA rhythmicity.

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Ribosome profiling (middle) and RNA (right) polar histograms for transcripts with an mRNA

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JTK-reported phase P > 0.05 are also shown. (E) Panther analysis of gene ontology

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biological processes based on the 2687 genes in (D). (F and G) There were 168 transcripts

9from (B) with a phase distribution between CT22-CT0 (F) corresponding to circadian rhythm

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and metabolism processes by Panther analysis (G). Note that this group includes NR1D1-

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regulated transcripts including Npas2, Arntl, Cry1, Nfil3, and Clock.

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Supplementary Figure 7 | Disruption of the Per2 uORF in mice (related to Figure 4). (A)3

An ssODN replaced the Per2 uORF (ATGTGA) with a mutant version (GTAGGT) in which

4

both the start and stop codons were mutated. The diagram shows the location of the CRISPR

5gRNA and upstream E'box within the Per2 promoter region (left). Representative Sanger

6

sequencing results from wild-type, Per2 uORF mutant, and heterozygous mice are shown7(right).

16

1
2

Supplementary Figure 8 | Detailed analysis of 12 h light:12 h dark (LD) sleep

3

parameters (related to Figure 4). (A) Mean sleep duration over 24 h averaged over 13 d for4

male (left) and female (right) mice. Red, Per2 uORF mutant mice. Black, wild-type. P

5values, Student's unpaired t-test. (B) Sleep episode duration (left) was shorter and Psw was

6higher (right) in Per2 uORF mutant mice than in wild-type mice, but the differences were not17

1

significant. (C) There were no significant differences in amplitude (left), wake episode2

duration (middle), and Pws (right) between Per2 uORF mutant and wild-type mice. (D and E)

3

Mean sleep duration per hour averaged over 13 d for 4-h windows from ZT4–8 (D) and ZT8–

412, ZT16–20, and ZT20–24 (E).

5

18

1
2

Supplementary Figure 9 | Analysis of mice in DD conditions, and cosinor analysis of LD

3

and DD circadian rhythm parameters (related to Figure 4). (A) Sleep duration per hour4over 12 d in constant darkness (DD) conditions for Per2 uORF mutant and wild-type male19

1

and female mice. Each row indicates data from one mice. (B) Sleep duration per hour over 24

2h in DD, averaged over 12 d for Per2 uORF mutant (red) and wild-type (black) male (top)3and female (bottom) mice. Lines indicate mean sleep duration at each time of day for each4

strain. Shaded area, SD at each time point. (C) Mean sleep (left) and wake (right) duration

5

over 24 h, averaged over 12 d. Red, Per2 uORF mutant mice. Black, wild-type. (D) Cosinor

6

analysis of the period (left) and amplitude (right) of wild-type and Per2 uORF mutant mice in

7

DD conditions. (E) Cosinor analysis of the amplitude of wild-type and Per2 uORF mutant

8

mice in LD conditions.

9

20

1
2

Supplementary Figure 10 | Comparison of RNA expression, translation, and protein

3

production phases in DD (this study) to reported phases from previously published4

studies in LD conditions (6, 8, 9). (A) Protein (orange) and mRNA (blue) phases for 16

5

selected circadian genes observed in DD (this study) or LD (6). (B) The delay between

6mRNA and protein phases observed in DD (this study) or LD (6). Several proteins (labeled)7

including PER2 had a greater than one hour difference in protein-mRNA delay between LD

8

and DD conditions. (C) The mRNA (left) and ribosome profiling (right) phases for 16

9selected circadian genes observed in DD (this study) or LD (8, 9). (D) The delay between

10mRNA and ribosome profiling phases observed in DD (this study) or LD (8, 9).

11

21

1
22

1

Supplementary Figure 11 | Extended FACS analysis of fluorescent reporter cells with2

and without the Per2 uORF (related to Figure 3). (A) Histogram (left) of GFP+ (top) and3

mCherry+ (bottom) cells from 3T3 cells transfected with pD1-P(Per2)-uORF-EGFP (-uORF,

4red) and pD1-P(Per2)-mCherry. (B) Histogram (left) of GFP+ (top) and mCherry+ (bottom)

5cells from 3T3 cells transfected with pD1-P(Per2)-EGFP (+uORF, black) and pD1-P(Per2)-

6mCherry. The backgating strategies for each gate in (A) and (B) are shown (right), including7

the percentage of GFP+ and mCherry+ cells in the population (rightmost graph). (C)8Scatterplot of GFP versus mCherry expression for -uORF (red) and +uORF (black) cells

9gated on GFP+ expression. (D) Histogram of the GFP/mCherry ratio for cells in (C).

10

23

1
24

1

Supplementary Figure 12 | Detailed analysis of the Per2 uORF in PER2:LUC MEF

2cells. (A) Bioluminescent recordings of wild-type PER2:LUC MEF cells (black) or

3heterozygous PER2:LUC MEF clones containing a mutated Per2 uORF (ATGTGA to

4

GTAGGT, red). Each graph is an independent experiment performed on a different day;5

multiple traces on the same graph indicate different clones. (B) Average bioluminescence

6

expression between 24 h and 72 h after dexamethasone stimulation of wild-type (black) and7

Per2 uORF mutant (red) MEFs.

25

1
2

Supplementary Figure 13 | Multiple sequence alignment of the Per2 5'UTR. (A) Multiple

3

sequence alignment of selected Per2 5'UTRs from different mammalian species using

4

MAFFT (10). Identical nucleotides (asterisk) or pyrimidine or purine nucleotide conservation

5(period) among all species is shown. Note: only Per2 transcriptional isoforms from each6species containing the Per2 uORF were used for the alignment.

7

26

1
2

Supplementary Figure 14 | Analysis of the IRES in the Nrd1 3'UTR and distribution of3near-cognate start codons and stop codons in selected 5'UTRs. (A) Extensive ribosome

4binding in Nr1d1 5' UTR overlaps with the region (orange) necessary for IRES-mediated5translation of Nr1d1 (11). (B) The efficiency of IRES-mediated translation for the Nr1d1

6

5'UTR and Cry1 5'UTR relative to a known IRES in Bcl2 5'UTR (12). (C) The 5' UTRs of

7

Dbp, Per3, Clock, and Cry1 have extensive ribosome binding in regions without a cognate

8

uORF. Cognate and near-cognate start codons (light blue) and stop codons (orange) indicate

9

regions of possible canonical uORFs (uORFs with an AUG start codon, shaded regions) and10

near-cognate uORFs (uORFs with a CUG, UUG, GUG, or ACG start codon).

11

27

1
2

Supplementary Figure 15 | Circadian rhythmicity analysis of reporter cells with

3variable numbers of uORFs. (A) Construction of pGL3-P(Per2)-dLuc reporter plasmids

4

with variable number of uORFs. (B) Bioluminescent recording of 3T3 cells transfected with5

the Per2 short promoter containing exactly one uORF, 42 nt (1x uORF, black) or 6 nt (1x281rev. pos. uORF, red) from the luciferase CDS. (C) Bioluminescent recording of 3T3 cells

2transfected with the Per2 short promoter without an uORF (black) or containing (red) two

3

uORFs (left), three uORFs (middle), or four uORFs (right). Cosinor analysis of the PMT

4

traces in (B) and (C) showing amplitude (D), mesor (E), and period (F). (G) Bioluminescent5

recording of 3T3 cells transfected with the pGL3-P(SV40)-3xE'box-dLuc containing exactly

6

one uORF, 42 nt (1x uORF, black) or 6 nt (1x rev. pos. uORF, red) from the luciferase CDS.

7(H) Bioluminescent recording of 3T3 cells transfected with pGL3-P(SV40)-3xE'box-dLuc8

without an uORF (black) or containing (red) two uORFs (left), three uORFs (middle), or four9

uORFs (right). Cosinor analysis of the PMT traces in (G) and (H) showing amplitude (I),10

mesor (J), and period (K).

11

29

1
2

Supplementary Figure 16 | Relationship between the number of uORFs and translation3

efficiency (TE) in the mouse liver using RNA-seq data from (13) and ribosome profiling30

1data from this study. (A) Scatterplot of TE (mRNA RPKM from (13) divided by ribosome

2

profiling RPKM) versus transcript abundance (RPKM) colored by the number of uORFs in

3each transcript. The distribution (top) and mean (bottom) of transcripts with the4corresponding number of uORFs is shown for TE (B) and ribosome binding (C). Histograms

5of TE (D) and ribosome binding (E) for transcripts without an uORF (orange) or with one or6more uORFs (blue).

31

1
2

Supplementary Figure 17 | Extended analysis of translation efficiency (TE) in the mouse3

liver using RNA-seq data from (13) and ribosome profiling data from this study. (A)4Scatterplot of TE (mRNA RPKM from (13) divided by ribosome profiling RPKM) versus

5transcript abundance (RPKM) colored by uORF length for each transcript with exactly one

32

1

uORF. Scatterplot of TE (B) and ribosome binding (C) versus uORF length. Scatterplot of2

TE (D) and ribosome binding (E) versus distance of uORF to start of the CDS.

3

33

1
2

Supplementary Figure 18 | Validation of ribosome profiling libraries from wild-type3

and Per2 uORF mutant mice liver at ZT02-04 (related to Figure 4). (A) Twelve to4fourteen-week-old male wild-type and Per2 uORF mutant mice under LD conditions were

5sacrificed at ZT02-ZT04 and liver sections were snap-frozen for ribosome profiling, total

6

RNA sequencing, qPCR, and western blot analyses. (B) Read distribution within 5'UTRs,

7

CDS, and 3'UTRs compared with the size of those features. Ribosome profiling reads are8enriched for the CDS and 5'UTR, and relatively few reads map to the 3'UTR. (C) Frame

9

analysis of ribosome profiling reads of single protein isoform mRNAs shows a preference for10

frame 1 (the coding frame). Box-and-whisker plots: midline, median; box, 25th and 75th

11

percentiles. Whiskers are minimum and maximum values. (D) Box and whisker plots show

12

the distribution of read lengths from all sequences. The majority of reads were between 27

13

and 33 nt, which matches the footprint size of the ribosome. (E) Summary of mapped reads.
341(F) Pairwise correlation of ribosome profiling RPKM between wild-type replicates and2

between Per2 uORF mutant replicates shows a high degree of correlation in ribosome

3

binding between samples.

4

35

1
2

Supplementary Figure 19 | Ribosome profiling, RNA sequencing, qPCR, and western3

blot analysis of liver from wild-type and Per2 uORF mutant mice at ZT02-04 (related to4

Figure 4). (A) Western blot of PER2 and actin protein of total protein lysate from wild-type5(+ uORF) and Per2 uORF mutant (- uORF) mice liver. Expression analysis of Per2 mRNA36

1

from wild-type and Per2 uORF mutant mice liver by qPCR (B) and total RNA sequencing

2(C). Ribosome binding (RPKM) across the entire Per2 transcript (D) or compartmentalized3

by the 5'UTR, CDS, and 3'UTR (E). (F) Ribosome profiling reads in wild-type and Per2

4

uORF mutant mice in the 5'UTR. Note the absence of reads across the uORF in both samples

5

and an increased number of reads at the start codon in the Per2 uORF mutant mice. (G)6Translation efficiency (ribosome profiling RPKM divided by total RNA RPKM) in wild-type

7

and Per2 uORF mutant mice. (H) Differential expression analysis by DESeq2.0 (14)8indicating genes with a significant change in gene expression as defined by an adjusted p-

9value < 0.05 and an absolute log2 fold change  0.58 (blue) shows that Per2 mRNA is

10significantly increased in Per2 uORF mutant mice. (I) Immunoblot analysis of PER2, actin,

11

and histone H3 of cytoplasmic and nuclear lysates from wild-type (+ uORF) and Per2 uORF

12

mutant (- uORF) mice liver.

13

37

1
38

1

Supplementary Figure 20 | Uncropped western blot images from Fig. S19A. (A) Cropped

2

western blot reproduced from Fig. S19A. Original uncropped images of the PER2 (B) and3

actin (C) western blot (top) and brightfield image showing the molecular weight ladder4(bottom). Cropped region (black boxes).

5

39

1
40

1

Supplementary Figure 21 | Uncropped western blot images from Fig. S19I. (A) Cropped

2

western blot reproduced from Fig. S19I. Original uncropped western blot of PER2 (B) with3

the molecular weight ladder visible in the outermost lanes. Uncropped images of actin (C)4and histone H3 (D) western blot (top) and brightfield image showing the molecular weight5

ladder (bottom). Cropped region (black boxes).

41

1

Table S1 | Sequence statistics for ribosome profiling data showing the circadian time2(CT), number of reads per sample, number of reads mapped to rRNA, unmapped3

reads, and mapped reads.
rRNA

rRNA

aligned(%)

67420894

31637068

4764936651029290161

43

479568956

40132748

50

6120169833316039

42

A3

8

92923717

46040656

50

7305061839578000

43

A4

12

89178791

37939665

434558237546680889

52

A5

16

73866781

37993296

51

3002837432870648

44

A6

20

57310601

29343482

5137852097

24181910

42

A7

24

57919810

30413247

53

1903635

325602928

44

B1

0

82543812

46972438

575223484630347890

37

B2

4

76767842

45784936

60

2978293428004613

36

B3

8

67206206

3192142947

2040538333244239

49

B4

12

77788251

44039811

57

26327013

31115739

40

B5

16

73477498

42486921

58

2420565328570012

39

B6

20

79546292

41792698

53

4794899632958695

41

B7

24

91267817

37921444422054622823

32800145

36

1066787268

544419839

51

73805521

7448561908

42

Sample

CT

Reads

A1

0

A2

Total

Unmapped

Unmapped(%)

MappedMapped
(%)

4

42

1

Table S2 | JTK analysis of ribosome profiling data for circadian (BH.Q < 0.05) transcripts.
Name
Sigmar1
Tardbp
Aqp8
Rdh11
Arntl
Ncald
Ppard
Rnf144aDennd2d
Pnrc1
Aqp8
Pgd
Cks2
Capn2
Tjp2
Wee1
Clpx
Nampt
Pmvk
Nat8
Evi5
Dpp9
Tjp3
Bloc1s5
Gpcpd1
Dock6
Fam222aElovl3
Hacl1
Cyp8b1
Adck3
Pnp
Dhcr7
Efna1
Nr1d1
Caprin1
Gale
Avpr1aAsb13
Mmab
RfxankLgalslChka
Camk1d
Per2
St5
Dus2
Slc34a2
Ccdc151
Cyb5b
Eif4ebp3

RefSeq ID
NM_001286541
NM_145556
NM_001109045
NM_021557
NM_001243048
NM_001170866
NM_011145
NM_001081977
NM_001093754
NM_001033225
NM_007474
NM_001081274
NM_025415
NM_009794
NM_001198985
NM_009516
NM_001044389
NM_021524
NM_026784
NM_023455
NM_007964
NM_172624
NM_001282095
NM_139063
NM_001042672
NM_177030
NM_001004180
NM_007703
NM_019975
NM_010012
NM_001163290
NM_013632
NM_007856
NM_001162425
NM_145434
NM_001111290
NM_178389
NM_016847
NM_178283
NM_029956
NM_001025589
NM_173752
NM_013490
NM_177343
NM_011066
NM_001001326
NM_025518
NM_011402
NM_001163787
NM_025558
NM_201256

BH.Q
0.00
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03

ADJ.P
1.23E-07
5.99E-07
2.20E-06
2.20E-06
2.20E-06
2.20E-06
2.20E-06
2.20E-06
2.20E-06
6.89E-06
6.89E-06
6.89E-06
6.89E-06
6.89E-06
6.89E-06
6.89E-06
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
1.89E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
4.69E-05
1.07E-04
1.07E-04

PER
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

LAG
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
16
0
18
0
22
0
0
0
0
12
0
0
1
0
2
0
0
0
0
10
0
0
0
0
0
0
0
0
2
18
0
2
0
20
0
18

AMP
8.1
1.2
6.5
5.6
2.2
1.7
1.0
0.6
0.3
25.7
21.1
5.6
3.4
2.0
1.2
1.1
26.1
11.4
6.8
4.2
2.5
2.0
1.8
1.6
1.5
0.8
0.5
182.4
77.4
60.6
44.8
11.6
10.3
8.6
7.0
6.3
5.1
5.0
4.7
3.0
2.7
2.6
2.5
1.2
1.2
0.8
0.6
0.6
0.3
50.9
43.8

CT00A
36.2
4.9
26.2
27.3
4.7
5.0
5.9
2.9
1.7
59.5
98.7
21.4
28.2
11.3
5.2
0.6
67.5
14.6
40.6
7.1
11.3
12.0
5.5
13.6
3.0
3.2
2.4
634.0
247.4
250.8
192.7
39.3
75.9
44.0
1.3
30.6
21.9
22.8
20.5
11.5
9.0
10.8
7.7
6.6
2.8
2.1
3.5
5.4
0.7
283.6
97.9

CT00B
32.8
4.2
22.8
21.1
4.1
4.6
4.9
4.1
1.4
68.6
86.8
22.5
22.4
12.4
4.0
0.6
67.5
13.2
32.2
9.3
13.3
10.9
5.4
11.4
2.4
2.7
3.4
590.9
188.7
224.1
178.5
43.4
59.3
29.9
1.3
24.1
16.3
20.0
20.2
10.0
7.4
6.4
6.3
4.2
1.5
2.6
2.6
3.0
0.7
239.2
75.0

CT04A
15.6
2.3
12.4
12.2
2.0
5.7
0.5
1.3
0.9
43.2
55.5
14.4
13.1
7.7
3.3
0.1
57.7
6.6
9.7
5.8
9.8
7.5
3.8
8.2
3.8
1.9
0.4
326.6
134.6
172.1
142.5
33.2
33.6
25.3
20.9
19.0
9.8
12.6
14.3
7.5
5.7
5.0
4.8
3.8
0.7
1.9
2.5
0.6
0.6
169.2
30.9

CT04B
16.6
1.7
7.4
9.5
2.4
5.5
1.4
1.3
0.9
40.4
31.4
8.9
11.4
6.4
2.4
0.3
48.2
6.9
7.2
4.3
8.5
6.9
2.0
7.8
3.2
1.2
1.1
365.0
114.1
160.4
97.0
29.3
30.7
25.9
8.1
13.9
8.2
10.7
11.4
5.1
6.7
4.4
5.0
3.3
0.2
1.4
2.5
0.8
0.3
130.8
24.6

CT08A
8.7
1.1
3.4
7.5
0.0
3.5
0.3
0.6
0.8
16.3
17.2
8.5
6.1
4.6
1.6
2.0
18.4
7.4
4.6
2.7
5.6
4.7
1.0
6.6
9.1
0.8
0.7
49.3
75.0
92.1
66.5
14.7
23.2
17.4
32.1
10.1
2.8
7.8
7.7
5.1
2.2
1.5
1.3
2.3
1.3
0.8
0.7
0.0
0.1
106.1
53.1

CT08B
9.2
1.0
3.1
9.4
0.3
3.4
0.3
0.5
0.6
11.6
12.9
9.1
8.6
4.8
1.9
1.0
16.1
9.3
5.3
3.3
5.5
4.0
1.3
5.5
4.8
0.8
0.4
64.1
64.3
75.7
36.9
16.0
29.5
19.4
18.8
9.5
5.1
5.0
7.4
3.5
2.5
1.2
1.5
2.6
1.2
0.7
1.8
0.2
0.3
82.4
18.2

CT12A
3.7
0.3
1.0
4.1
0.0
2.1
0.0
0.0
0.4
11.1
7.7
7.1
2.4
4.3
1.6
2.3
9.1
31.1
4.2
3.3
5.1
2.4
0.7
4.0
23.0
0.5
0.0
27.1
46.6
47.1
24.3
13.6
24.1
9.0
6.8
7.6
1.9
3.0
5.5
3.0
2.4
0.7
0.2
2.5
2.0
0.3
1.5
0.0
0.7
75.6
187.2

CT12B
7.3
1.0
1.4
3.3
0.1
1.1
0.1
0.2
0.4
8.5
6.3
6.7
6.7
3.0
1.0
2.1
5.6
22.1
4.3
3.1
2.6
3.4
0.6
5.5
11.7
0.4
0.2
24.1
42.3
51.5
21.4
8.5
13.9
12.3
9.2
5.9
2.2
1.6
5.7
3.0
1.9
0.5
0.5
0.9
2.0
0.3
1.4
0.0
0.3
67.5
100.8

CT16A
13.5
1.9
4.3
9.5
1.5
2.4
1.1
1.0
1.0
34.7
19.0
11.9
10.1
6.1
3.1
3.8
22.4
43.9
8.9
8.4
7.9
5.9
3.5
7.8
4.5
1.4
0.9
99.2
140.7
74.6
70.3
26.6
25.5
24.4
5.6
18.6
8.2
9.8
12.3
5.2
5.7
5.5
3.6
0.8
6.6
1.3
2.0
1.1
1.6
149.0
396.5

CT16B
9.7
2.0
2.3
7.6
1.3
2.0
0.6
0.7
0.9
16.5
15.4
13.7
9.6
6.6
2.2
2.9
17.0
32.4
9.5
7.6
6.0
4.9
1.8
6.5
4.3
1.4
0.7
40.9
84.9
80.9
44.7
20.4
34.6
15.1
2.0
15.5
10.9
6.2
8.4
4.3
3.6
3.2
2.0
1.2
4.8
1.0
1.8
0.2
0.8
97.2
111.1

CT20A
20.2
2.4
14.2
14.7
2.6
3.3
2.8
1.9
1.3
34.4
37.2
15.2
12.4
6.5
2.6
1.2
29.7
18.2
18.5
10.2
6.7
6.2
3.2
7.0
3.0
1.6
1.2
209.9
147.7
122.2
69.5
18.6
33.5
31.6
0.7
17.9
12.4
7.3
10.9
7.8
6.7
5.2
2.3
2.9
3.2
1.4
2.2
0.4
0.7
126.9
92.3

CT20B
22.8
2.7
11.4
20.0
3.8
3.4
2.1
1.0
1.0
44.1
49.7
17.2
10.5
7.4
3.5
1.3
46.0
20.5
24.3
9.8
8.9
6.6
3.2
8.8
2.9
2.0
1.4
318.8
199.3
134.5
100.3
27.3
49.8
27.2
0.8
18.0
11.0
12.1
12.6
7.3
6.4
6.7
4.6
2.8
2.8
1.9
2.2
1.3
1.0
148.2
110.7

CT24A
30.2
3.9
17.9
19.6
3.7
5.1
5.5
1.7
1.3
44.1
61.4
18.2
16.3
9.4
3.7
0.7
58.6
13.3
23.0
8.8
9.1
10.5
4.4
10.0
2.1
2.3
2.2
529.0
194.5
207.2
143.2
33.3
52.2
41.9
1.5
22.7
12.1
16.3
16.1
5.6
9.3
7.2
6.3
6.1
2.1
2.2
3.8
2.3
0.7
194.2
56.6

CT24B
21.9
3.1
17.4
16.1
3.0
5.1
3.1
1.9
1.5
49.7
51.7
16.0
13.9
7.9
3.3
0.4
63.6
10.9
16.8
8.5
9.9
7.1
4.0
8.6
1.9
1.7
1.2
450.4
180.9
165.3
123.5
34.5
52.5
26.6
0.8
22.9
10.9
14.8
12.9
9.1
7.8
6.7
5.8
5.1
1.6
1.5
3.4
2.6
0.7
173.6
64.6

43

Gys2
Nop10
Mrap
Acss2
Ppp1r3c
Sgpl1
Hypk
Pter
Tfpi2
Slc16a1
Tgm2
Btg1
Cdkn1aSpryd7Acacb
Pi4k2a
Lsr
Ddx17
Ext2
Ncoa5
Atg7
Slc17a8
Agpat5
Rbmx
Dut
Mthfr
Per3
Oma1
Mtrf1
St5
Plekhg1
Eif2ak4
Creb3l1
Prdm10
Bhmt
Plin2
S100a10
Cyb5r3
Sdc1
Tubb2a
Ddc
Gclc
Tars
Pnkd
Hmgcs1
Cldn2
Acsl4
Adh4
Ran
Fdft1
Mal2
Atg1012610528J11Rik
Lpin1
Ubqln1
Nmrk1

NM_145572
NM_025403
NM_029844
NM_019811
NM_016854
NM_009163
NM_026318
NM_008961
NM_009364
NM_009196
NM_009373
NM_007569
NM_001111099
NM_025697
NM_133904
NM_145501
NM_001164185
NM_001040187
NM_010163
NM_144892
NM_001253717
NM_182959
NM_026792
NM_001166623
NM_023595
NM_001161798
NM_011067
NM_025909
NM_145960
NM_029811
NM_001033253
NM_013719
NM_011957
NM_001080817
NM_016668
NM_007408
NM_009112
NM_029787
NM_011519
NM_009450
NM_001190448
NM_010295
NM_033074
NM_001039509
NM_145942
NM_016675
NM_001033600
NM_011996
NM_009391
NM_010191
NM_178920
NM_026566
NM_025572
NM_001130412
NM_152234
NM_145497

0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
1.07E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

16
0
0
0
1
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
16
0
0
2
0
0
0
0
0
2
0
0
0
0
0
2
0
0
0
0
20
2
0
0
0
0
0
15
0
18

22.4
19.1
13.4
12.6
10.7
7.1
7.0
5.8
5.8
5.5
5.4
5.1
3.9
3.7
3.7
3.4
2.8
2.3
1.5
1.0
0.9
0.9
0.9
0.8
0.5
0.4
0.4
0.3
0.3
0.2
0.1
0.1
0.1
0.1
57.1
46.8
42.9
35.4
33.1
29.5
19.3
16.2
15.6
12.5
11.2
11.0
10.4
9.8
8.6
6.2
5.7
5.4
4.9
4.3
3.9
3.9

45.8
197.0
54.0
80.3
77.1
30.3
62.3
28.1
34.1
33.5
58.5
16.9
22.0
20.5
17.5
17.6
14.7
17.6
9.1
5.3
6.0
3.3
6.0
4.0
5.5
1.6
0.1
8.6
2.8
0.3
0.8
0.5
0.4
0.5
362.2
236.4
251.9
317.2
203.0
79.5
81.0
66.9
62.1
46.8
62.0
49.0
31.2
57.5
63.1
22.9
24.9
28.0
16.4
3.8
29.4
6.0

41.6
170.9
47.0
62.4
39.5
22.3
58.0
24.0
31.4
25.6
37.5
16.3
14.0
16.4
13.0
16.8
11.8
10.2
10.2
3.9
5.5
3.1
7.4
2.9
4.2
1.0
0.2
7.6
2.4
0.4
0.8
0.6
0.6
0.6
255.4
188.9
240.6
278.3
165.9
60.2
59.3
71.1
52.9
41.1
58.9
42.0
26.2
58.3
52.5
21.4
24.0
24.8
16.6
3.6
29.1
5.5

31.0
123.8
33.6
36.2
25.9
21.8
28.7
22.0
21.1
16.4
25.8
11.4
4.5
13.5
6.8
14.7
5.8
8.4
5.9
2.3
4.1
2.2
3.7
2.3
2.2
1.0
0.0
5.2
2.0
0.1
0.5
0.2
0.2
0.3
289.4
145.4
210.9
210.4
96.1
51.5
45.5
60.3
43.9
32.1
37.6
35.3
18.7
51.4
34.4
7.1
11.4
20.2
13.3
2.7
21.9
3.5

21.7
122.5
24.9
21.1
24.0
15.8
33.7
17.4
20.7
14.3
20.6
7.9
5.7
9.2
4.8
10.8
7.9
6.9
4.8
1.9
3.4
1.1
4.2
1.5
2.5
0.9
0.0
5.1
2.1
0.2
0.6
0.2
0.2
0.4
185.9
159.6
169.6
187.7
85.7
36.8
47.0
51.2
33.1
22.8
26.4
31.6
17.0
43.1
25.9
7.5
9.4
16.7
9.9
1.9
19.8
4.3

58.8
107.9
16.4
23.1
12.0
13.2
31.7
13.2
13.0
10.0
18.1
6.0
0.4
8.6
2.8
8.4
4.8
4.8
4.6
1.4
2.8
0.9
3.0
1.4
2.6
0.2
0.5
5.1
1.4
0.1
0.4
0.1
0.1
0.4
124.4
137.3
110.4
184.2
60.3
5.7
18.4
44.5
20.1
14.5
23.5
20.9
14.2
43.5
21.7
6.6
5.8
12.8
10.3
7.2
16.6
3.9

36.8
102.6
14.5
16.3
11.7
11.6
27.0
15.0
16.8
11.4
16.3
5.1
2.3
8.2
2.7
8.1
5.3
5.1
3.7
1.3
2.5
0.5
3.4
1.5
2.0
0.2
0.4
4.9
1.2
0.1
0.3
0.1
0.1
0.3
119.1
93.5
111.9
160.3
47.8
9.8
22.2
29.1
18.8
9.8
41.8
19.7
16.6
42.5
23.6
13.6
4.8
11.9
5.5
7.3
14.5
3.4

85.5
86.0
12.6
18.3
11.1
9.9
19.0
10.6
12.2
8.3
13.1
3.6
0.5
7.2
3.9
5.6
4.2
4.5
3.1
0.9
2.1
0.3
1.4
0.7
0.7
0.0
1.4
3.3
1.9
0.0
0.5
0.1
0.1
0.2
114.4
56.2
94.9
157.6
63.9
4.8
13.2
26.7
14.3
9.1
16.9
14.2
23.7
32.3
20.5
6.4
4.2
7.2
5.4
66.9
14.1
14.9

70.9
95.2
12.1
13.5
4.3
6.8
22.6
7.3
8.3
8.2
10.9
2.8
0.6
7.5
2.7
7.3
4.7
3.4
3.0
1.0
2.2
0.9
2.5
1.1
1.1
0.1
0.8
2.6
1.2
0.0
0.3
0.1
0.1
0.2
86.9
66.7
66.3
146.6
46.1
2.4
12.9
23.5
12.7
6.7
10.5
16.8
16.2
22.4
21.5
4.2
6.9
8.3
3.6
50.2
12.6
9.5

103.3
188.9
35.3
26.8
18.4
19.6
34.5
16.9
17.0
20.8
24.7
9.5
6.6
14.5
5.9
13.7
10.8
7.8
5.9
3.5
4.0
1.3
5.1
2.2
2.0
0.8
1.0
6.6
1.9
0.1
0.5
0.3
0.3
0.3
175.5
118.5
153.0
258.5
144.9
37.9
54.8
35.5
35.3
30.0
25.0
32.8
31.7
42.4
36.9
10.7
22.2
18.9
9.4
17.8
28.3
16.2

86.3
115.7
19.3
25.7
9.5
17.6
33.4
12.5
14.4
14.6
24.1
3.3
2.6
9.2
4.7
9.4
6.2
7.0
3.4
2.0
3.4
1.4
4.1
1.1
2.0
0.4
0.9
4.9
1.7
0.0
0.5
0.2
0.2
0.3
136.1
90.0
110.9
188.5
71.1
17.9
37.3
28.3
27.6
14.6
21.7
20.6
35.1
37.5
28.7
11.3
9.2
11.0
6.4
15.8
18.5
11.6

40.5
129.7
35.4
30.0
19.5
15.9
42.5
15.9
12.3
14.5
24.2
6.3
7.8
15.0
8.5
10.5
9.5
7.4
5.8
2.1
3.5
2.2
4.0
2.3
5.4
0.5
0.5
5.4
2.1
0.4
0.6
0.5
0.2
0.4
138.8
106.9
128.9
190.8
107.8
27.2
43.1
31.7
26.4
21.8
31.3
19.7
30.2
30.9
26.9
15.4
14.9
13.9
7.9
5.1
21.0
7.0

53.7
135.0
28.4
58.4
38.5
21.8
36.9
20.7
25.8
20.8
23.2
12.3
5.7
12.4
13.7
12.0
8.1
8.2
5.2
2.8
3.1
1.7
4.6
2.7
2.6
0.8
0.3
6.0
2.0
0.2
0.6
0.3
0.3
0.4
248.3
141.7
160.6
233.0
105.6
39.2
56.8
44.7
40.9
25.1
56.0
37.2
38.0
51.7
33.8
17.9
13.8
17.0
11.6
4.0
21.0
7.2

39.0
147.9
33.3
53.5
62.1
24.8
39.9
28.6
27.0
21.6
35.4
12.6
7.6
14.7
12.7
15.6
11.9
13.3
6.6
3.6
4.8
2.3
5.4
3.2
3.3
1.6
0.2
6.4
2.3
0.4
0.7
0.4
0.3
0.5
245.3
195.0
191.6
253.0
129.4
83.6
52.4
63.3
49.9
35.1
57.4
38.8
29.8
58.5
50.7
19.6
17.8
22.8
13.5
3.4
25.9
4.5

36.6
135.6
41.7
48.8
56.4
25.0
35.7
21.5
29.7
21.1
27.5
13.5
12.4
13.8
10.7
14.0
10.2
11.5
7.0
4.1
4.4
1.7
5.7
2.5
3.1
1.2
0.2
6.0
2.5
0.4
0.7
0.3
0.3
0.4
318.2
145.1
193.6
235.9
117.2
51.2
55.4
54.3
40.4
26.7
54.0
35.5
27.1
64.0
38.4
18.6
18.0
21.1
14.1
2.8
23.5
5.5

44

Anp32a
Ppm1b
Tnfaip2
Tlcd1
Pabpn1
Tspan33
Acnat1
40057
Lpin2
Mtap
Taf15
Rpa1
Lnx2
Gca
Bco2
Itgb5
Sorbs2
Arntl
40787
Shkbp1
Syne1
Rad9a
Smarcal1
Gtf3c5
Ptpn9
Ddhd1
E4f1
Sorbs2
Opn3

NM_009672
NM_001159496
NM_009396
NM_026708
NM_019402
NM_146173
NM_001164565
NM_001113487
NM_022882
NM_024433
NM_027427
NM_001164223
NM_080795
NM_145523
NM_133217
NM_001145884
NM_001205219
NM_007489
NM_001009818
NM_138676
NM_001079686
NM_011237
NM_018817
NM_148928
NM_019651
NM_001039106
NM_007893
NM_172752
NM_010098

0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05

2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04
2.29E-04

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

0
0
1
18
0
0
0
0
14
0
0
0
0
0
0
0
0
0
0
0
4
22
0
0
0
0
0
0
0

3.4
2.9
2.8
2.7
2.6
2.3
2.1
2.1
2.1
2.0
1.7
1.5
1.4
1.2
1.1
0.9
0.9
0.9
0.7
0.7
0.5
0.5
0.4
0.4
0.3
0.3
0.3
0.2
0.1

39.8
16.6
10.6
7.5
15.8
9.3
11.9
11.8
4.4
16.0
17.6
11.6
7.3
3.6
6.2
8.4
5.1
2.3
5.0
2.7
1.4
1.4
3.5
3.9
3.3
2.1
1.3
2.2
0.8

39.6
16.0
8.3
4.4
14.0
8.4
10.6
10.4
4.9
14.0
14.4
10.2
6.8
5.4
6.3
6.8
4.3
2.9
4.3
2.3
1.8
1.1
3.3
2.5
2.3
1.9
0.9
2.6
0.6

29.9
13.9
7.3
4.9
10.4
7.2
7.1
7.7
3.6
7.4
8.6
8.1
3.8
2.9
4.2
4.1
3.9
2.1
3.2
1.7
2.1
0.6
2.3
1.8
1.3
0.9
0.6
1.8
0.1

21.8
9.3
6.6
3.9
8.2
5.7
6.3
6.8
3.8
7.6
6.8
6.3
3.7
2.5
3.3
4.5
3.9
1.2
2.3
1.5
1.5
0.6
2.1
1.8
1.6
1.0
0.6
1.4
0.2

20.0
10.3
4.0
6.0
7.5
4.6
4.9
5.2
6.8
6.5
6.1
5.0
2.1
1.3
4.1
3.4
3.0
0.2
1.9
1.1
1.1
0.1
1.7
1.4
0.8
0.6
0.4
1.4
0.1

17.0
7.2
2.8
5.0
6.8
3.7
3.7
4.3
5.6
6.1
7.2
6.2
2.1
1.5
2.7
2.8
3.2
0.2
2.2
0.9
0.8
0.2
1.5
1.4
1.1
0.6
0.5
1.6
0.2

19.8
8.3
2.5
8.1
5.5
3.6
3.3
3.3
10.9
5.6
8.0
5.4
1.3
0.1
1.9
3.2
2.0
0.3
1.6
1.0
0.6
0.3
0.9
0.7
1.0
0.8
0.4
0.9
0.0

15.4
5.6
1.4
6.4
5.6
3.1
2.3
2.3
8.6
4.8
5.6
4.6
1.5
1.2
1.7
2.2
1.7
0.0
1.1
0.9
0.3
0.3
1.0
1.1
1.1
0.5
0.3
0.9
0.0

21.0
12.5
4.3
10.6
12.6
5.6
7.8
7.9
8.9
11.2
9.2
6.0
6.5
2.3
3.0
4.8
4.6
0.7
2.4
1.7
0.6
0.8
2.0
1.6
1.9
1.4
0.8
1.7
0.3

20.0
8.9
2.6
8.7
7.6
3.5
6.8
6.4
7.8
6.1
8.6
6.9
3.2
2.3
2.8
3.4
3.3
0.8
4.4
1.1
0.5
0.7
2.1
1.4
1.1
0.8
0.5
1.5
0.1

17.3
11.1
4.7
9.0
9.5
6.7
4.9
8.1
4.9
7.8
9.6
6.0
4.1
3.1
2.9
3.5
4.1
1.0
3.2
2.1
0.6
1.0
1.9
1.4
1.3
0.9
1.0
1.5
0.2

31.3
11.7
5.7
8.4
13.2
5.0
7.9
7.7
5.4
10.2
10.4
7.7
3.4
2.5
4.6
4.8
4.8
1.5
3.3
1.6
1.0
1.0
2.1
2.2
2.3
1.1
0.8
2.0
0.3

37.0
15.7
8.0
7.1
13.2
7.0
10.9
9.1
4.0
10.1
13.6
9.5
5.9
2.0
5.8
6.4
5.0
2.1
3.4
2.0
1.8
1.3
2.8
2.4
2.1
1.5
1.1
2.0
0.4

32.9
14.5
6.4
6.2
8.6
8.0
8.0
7.3
2.9
13.9
9.6
8.3
4.3
4.0
4.5
5.2
4.2
1.0
4.0
1.7
1.7
0.7
2.3
1.7
2.9
1.4
0.7
2.1
0.5

1

45

Table S3 | Sequence statistics for ribosome profiling data for wild-type (M1W,
M3W, M6W) and Per2 uORF mutant (M15, M20D, M21D) showing the number ofreads per sample, number of reads mapped to rRNA, unmapped reads, and mapped
reads.
rRNA

rRNAaligned(%)

31067707

13722226

44

34955021113849979

45

M3W

30481515

12873480

4232359111114372124

47

M6W

37574250

18179097

48

3539752915855401

42

M15D

38245244

1812817747

3624572916492495

43

M20D

42932671

19864776

4642121701018855725

44

M21D

33744849

145252194336014891115618141

46

SampleReads

M1W

Unmapped

Unmapped(%)

MappedMapped(%)

46

Dataset S1 (separate file). JTK analysis of ribosome profiling data for all transcripts.
Dataset S2 (separate file). JTK analysis of ribosome profiling data for all uORFs.
Dataset S3 (separate file). Translation efficiency using RNA-seq data from (13) andribosome profiling data from this study.
Dataset S4 (separate file). Ribosome profiling, total RNA sequencing, and translation

efficiency from the livers of three wild-type (M1W, M3W, and M6W) and three Per2
uORF mutant (M15D, M20D, and M21D) mice sacrificed at ZT02-04.
Dataset S5 (separate file). Differential expression analysis of total RNA from the livers of

three wild-type (M1W, M3W, and M6W) and three Per2 uORF mutant (M15D, M20D,
and M21D) mice sacrificed at ZT02-04.

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B. Langmead, S. L. Salzberg, Fast gapped-read alignment with Bowtie 2. Nat. Methods 9,357–359 (2012).

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F. A. Ran, et al., Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8,2281–2308 (2013).

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J. Wang, et al., Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse
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11.

D.-Y. Kim, K.-C. Woo, K.-H. Lee, T.-D. Kim, K.-T. Kim, hnRNP Q and PTB modulate thecircadian oscillation of mouse Rev-erb alpha via IRES-mediated translation. Nucleic Acids
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48