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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
anti-Per2
Doi et al., 201921
N/A
anti-Cry2
MBL
PM082
anti-Clock
MBL
D349-3
anti-Bmal1
MBL
D335-3
anti-HSF1
Abcam
Cat# Ab61382; RRID: AB_942016
Antibodies
anti-p-CREB
Cell Signaling
Cat# 9198; RRID: AB_2561044
anti-FosB
Cell Signaling
Cat# 2251; RRID: AB_2106903
anti-tubulin
Sigma
Cat# T6199; RRID: AB_477583
anti-b-actin
Sigma
Cat# A5441; RRID: AB_476744
AAV-DJ
Grimm et al., 200858
N/A
DH5a Escherichia coli
Takara Bio
#9057
Bacterial and virus strains
Chemicals, peptides, and recombinant proteins
17b-hydroxy-wortmannin
Cayman
#13812
PI3-Kinase a Inhibitor 2
Cayman
#21197
GSK1059615
Cayman
#11569
EGF
Peprotech
14321-54
Dexamethasone
Sigma
D8893
Cycloheximide
Nacalai Tesque
06741-91
Biotin-PEG4-alkyne
Sigma
#764213
KNK437
Sigma
SML0964
Rapamycin
Cayman
#13346
MK2206
Selleck chemicals
S1078
U0126
Cayman
#70970
DZnepA
Sigma
SML0305
Ruthenium Red
Wako
189-03181
Thermo Fisher
C10276
Ribo-seq and RNA-seq data (for Figures 1, 3, and S1)
This paper
GEO: GSE188529
Ribo-seq data (for Figures 3 and S1)
This paper
GEO: GSE211532
Unprocessed original immunoblot data
(for Figures 2, 3, 4, and S2–S6)
This paper
Mendeley Data: https://doi.org/10.17632/k4wygddpkd.1
Critical commercial assays
Click-iT Protein Reaction Buffer Kit
Deposited data
Experimental models: Cell lines
Per2::LucTS knock-in MEFs
This paper
N/A
Tet-On 3G NIH3T3
Clontech
#631197; RRID: CVCL_V360
This paper
RIKEN BRC No. 11461
Quantitative RT-PCR forward primer for Per2:
50 -CTC ACT GAG ATT CGG GAT ATG-30
Doi et al., 201921
N/A
Quantitative RT-PCR reverse primer for Per2:
50 -CTC CCA CCT TGT CTC CAG TC-30
Doi et al., 201921
N/A
Quantitative RT-PCR forward primer for Cirp:
50 -GCA GAT CTC CGA AGT GGT G-30
Morf et al., 20128
N/A
Experimental models: Organisms/strains
Per2 minimal uORF mutant mice
Oligonucleotides
(Continued on next page)
12 Cell Reports 42, 112157, March 28, 2023
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Article
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Quantitative RT-PCR reverse primer for Cirp:
50 -CAG CCT GGT CAA CTC TGA T-30
Morf et al., 2012
N/A
Quantitative RT-PCR forward primer for Rplp0:
50 -CTC ACT GAG ATT CGG GAT ATG-30
Doi et al., 201921
N/A
Quantitative RT-PCR reverse primer for Rplp0:
50 -CTC CCA CCT TGT CTC CAG TC-30
Doi et al., 201921
N/A
Probes and primers for Taqman qPCR, see Table S2
Doi et al., 2019,21
Yamaguchi et al., 202159
N/A
shRNA targeting sequence for control:
50 -ATA ACA TGG CCA TCA TCA AGG AGT TCA TG-30
This paper
N/A
shRNA targeting sequence for Cirp #1:
50 -CAG AGA CAG CTA TGA CAG TTA-30
Thermo Fisher
https://www.thermofisher.com/jp/ja/home/
life-science/rnai/synthetic-rnai-analysis.html
shRNA targeting sequence for Cirp #2:
50 -CGT CCT TCC ATG GCT GTA ATT-30
Thermo Fisher
https://www.thermofisher.com/jp/ja/home/
life-science/rnai/synthetic-rnai-analysis.html
Recombinant DNA
pHSE-Luc
Clontech
#K2049-1
pCRE-Luc
Clontech
#K2049-1
pSRE-Luc
Clontech
#K2049-1
pTRE3G-Luc
Clontech
#631168
STAR v2.7.0
N/A
https://github.com/alexdobin/STAR
Samtools v1.10
N/A
https://github.com/samptools/samtools
fp-framing, fp-transcript
N/A
https://github.com/ingolia-lab/RiboSeq
ImageJ
NIH
https://imagej.nih.gov/ij/
Prism 8
GraphPad
N/A
Software and algorithms
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Masao Doi
(doimasao@pharm.kyoto-u.ac.jp).
Materials availability
Per2 m-uORF mutant mouse line has been deposited to RIKEN BioResource Research Center (RIKEN BRC No. 11461, https://
knowledge.brc.riken.jp/resource/animal/). All other reagents generated in this study are available from the lead contact with a
completed materials transfer agreement.
Data and code availability
d Ribo-seq and RNA-seq data have been deposited at GEO and are publicly available as of the date of publication. Accession
numbers are GSE188529 and GSE211532. Unprocessed original immunoblot data have been deposited to Mendely Data are
available at: https://doi.org/10.17632/k4wygddpkd.1. All other data reported in this paper will be shared by the lead contact
upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell lines
The Tet-On 3G NIH3T3 cell line was purchased from Clontech (Mountain View, CA) and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL G418 at 37 C in a humidified 5% CO2 atmosphere. To
generate Per2::LucTS knockin MEF cells, we used a firefly luciferase containing the mutations T214A, I232A, F295L, and E354K
(LucTS).26 The LucTS coding sequence was inserted in frame before the endogenous stop codon of Per2 using CRISPR/Cas9
Cell Reports 42, 112157, March 28, 2023 13
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Article
technology. The gRNA sequence used was 50 -TGA GGT ATC ACA GAT TCC CG-30 . The edited genome sequence was verified by
PCR-Sanger sequencing. Cells were maintained in DMEM/Ham’s F-12 (1:1) supplemented with 10% FBS.
Mice
The C57BL/6J male mice 6–8 weeks old were purchased from Japan SLC (Shizuoka, Japan). Per2 m-uORF mutant mice were established at the Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, by
CRISPR/Cas9-based genome editing using C57BL/6J mouse zygotes. The sequences of gRNA and the template oligonucleotide
were 50 -TTT CCA CTA TGT GAC AGC GGA GG-30 and 50 -CAA TGG CGC GCG CAG GGG CGG GCT CAG CGC GCG CGG TCA
CGT TTT CCA CTT AAC AGC GGA GGG CGA CGC GGC GGC AGC GGC GCT ACT GGG ACT AGC GGC TCC G-30 , respectively.
The edited sequence was verified by Sanger DNA sequencing. All procedures for animal experiments in this study were conducted in
compliance with the Ethical Regulations of Kyoto University and performed under protocols approved by the Animal Care and Experimentation Committee of Kyoto University and the Institutional Animal Care and Use Committees of Osaka university.
Primary fibroblasts
Primary fibroblasts were isolated from the lung or skin of adult Per2 m-uORF mutant mice and control wild-type siblings, according
to a protocol previously established.60 Briefly, the tissue fragments were cut into 1 mm pieces and incubated in Blendzyme 3
(0.14 Wunsch units/mL, Roche, 11814176001)-containing DMEM/F12 medium at 37 C for 90 min. After centrifugation for
5 min 3 524 g, the pellet was resuspended in DMEM/F12 media supplemented with 15% FBS, and then incubated at 37 C, 5%
CO2. Cells used for experiments were between passages 2 and 4.
METHOD DETAILS
Temperature control
WTS was applied to cells by transferring the cell dish from a 35 C incubator to a 38.5 C-prewarmed incubator. The temperature of the
medium was measured using an electronic thermometer and verified to approach 38.5 C at 7–8 min after being transferred. Application of simulated body temperature was performed using a custom-modified convection-ventilated incubator box, in which the
temperature was controlled by a Peltier device and forced air circulation. This device enabled the programming and recording of temperature profiles in real time with an accuracy of 0.1 C.
Ribo-seq and RNA-seq analysis
We used Dex-synchronized MEF cells treated with or without WTS for 2 h. Cells were lysed with a buffer containing 20 mM Tris-HCl
[pH 7.5], 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 100 mg/mL cycloheximide (CHX). Following the digestion of
genomic DNA with DNase I (25 U/mL, Thermo Fisher Scientific), lysates were centrifuged at 20,000 3 g and the resultant supernatant was processed for library generation.61 For Ribo-seq, samples (10 mg RNA each) were digested with 20 U RNase (Epicentre)
at 25 C for 45 min, and the RNA fragments protected by ribosomes ranging 17–34 nt were gel-excised and ligated with linker oligonucleotides followed by rRNA depletion using Ribo-Zero Gold rRNA Removal Kit (Illumina). The resultant RNA fragments were
reverse transcribed with Photoscript II (NEB) and circularized using CircLigaseII (Epicentre). The circularized cDNA templates
were PCR amplified for 6 to 8 cycles using Phusion polymerase (NEB) and sequenced on an Illumina Hiseq X or HiSeq 4000 instrument. For RNA-seq, samples were subjected to library construction using TruSeq Stranded Total RNA Gold (Illumina) and sequenced
on HiSeq 4000 as described62; single-end reads (150 bp for experiment 1 and 50 bp for the others) were mapped to the mouse
genome (GRCm38/mm10) using STAR (version 2.7.0) and sorted and indexed using samtools (version 1.10). For filtering rRNA
and tRNA, STAR was used with rRNA and tRNA annotations downloaded from the UCSC table browser. Footprints ranging from
20–34 nt were used for further analysis. In Figure 3G, the minimal uORF was identified as NUG-stop (N = A, C, U or G). The A-site
location of the Ribo-seq footprints and 3-nucleotide periodicity, which is supplied to validate our Ribo-seq data quality (Table S1),
were calculated as described61 using the fp-transcript and fp-framing, respectively (https://github.com/ingolia-lab/RiboSeq).
Polysome profiling
Linear 15%–50% sucrose gradients were prepared using a BioComp Gradient Master (Biocomp Instruments). Sucrose was dissolved in 20 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 100 mM KCl, 2 mM DTT, 100 U/mL human recombinant RNase Inhibitor
(TOYOBO), and 100 mg/mL CHX. Cells were lysed in ice-cold buffer containing 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM
MgCl2, 1 mM DTT, 1% Triton X-100, and 100 mg/mL CHX. Following the digestion of genomic DNA with DNase I (25 U/mL, Thermo
Fisher Scientific), lysates were centrifuged at 20,000 3 g. The supernatants were loaded on top of the gradients and sedimented at
35,000 rpm in an SW41 rotor at 4 C for 2 h. After centrifugation, gradients were fractionated using the Gradient Master instrument
with continuous monitoring at A260. RNA in each fraction was extracted using TRIzol LS reagent (Thermo Fisher Scientific).
Immunoblotting
To minimize proteolysis of the endogenous Per2 protein, cells and tissues (dorsal ear skin and lung) were lysed immediately in
Laemmli buffer containing 1x cOmplete Protease Inhibitor cocktail (Roche Diagnostics), as described previously.21 Immunoblots
14 Cell Reports 42, 112157, March 28, 2023
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were performed using our standard method with affinity-purified anti-mPer2 rabbit polyclonal antibody (final concentration,
2 mg/mL)21 or commercially available antibodies against a-Tubulin (Sigma Aldrich, T6199, 1:1,000), b-actin (Sigma Aldrich, A5441,
1:1,000), Clock (MBL, D349-3, 1:1,200), Bmal1 (MBL, D335-3, 1:200), Cry2 (MBL, PM082, 1:200), HSF1 (abcam, ab61382, 1:200),
p-CREB (Cell Signaling, #9198, 1:1,000), and FosB (Cell Signaling, #2251, 1:1,000). The CHX chase assay was performed by adding
CHX (100 mg/mL) to the medium at Time 24. Where specified, cells were treated with 17b-hydroxy Wortmannin (Cayman chemical,
10 mM), PI3-Kinase a Inhibitor 2 (Cayman chemical, 10 mM), GSK1059615 (Cayman chemical 10 mM), or EGF (Peprotech, 10 ng/mL).
Labeling of de novo protein synthesis
Dex-synchronized cells were preincubated with a Met/Cys-free DMEM medium (Thermo Fisher Scientific) for 3 h before experiments.
[35S]Methionine (14.8 MBq/sample, Muromachi Kikai) was added to the medium at Time 0 and incubated for 2 h with or without WTS.
Cells were lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 1% Nonidet P40, 1% sodium deoxycholate)
containing 1x cOmplete Protease Inhibitor cocktail (Roche Diagnostics) and subjected to immunoprecipitation with anti-mPer2 polyclonal antibody21 or anti-Cry2 antibody (MBL, PM082). The immunoprecipitate was separated by SDS-PAGE and visualized with an
X-ray film (Kodak). For click-labeling experiments, azidohomoalanine (AHA) was added to the medium at Time 0 (final concentration,
50 mM) instead of [35S]Methionine. Cells were lysed in RIPA buffer containing 1x cOmplete Protease Inhibitor cocktail. The lysates
were conjugated with biotin-PEG4-alkyne (Sigma Aldrich) using the Click-iT Protein Reaction Buffer Kit (Thermo Fisher Scientific).
After removal of excess free biotin-PEG4-alkyne using PD SpinTrap G-25 columns (Cytiva), AHA-biotin-alkyne labeled proteins
were affinity-purified with streptavidin beads (Thermo Fisher Scientific). The precipitates were analyzed by immunoblotting using
the anti-mPer2 polyclonal antibody,21 anti-Cry2 (MBL, PM082), or anti-b-actin antibodies (Sigma Aldrich, A5441). Biotin-labeled proteins were detected using Vectastain Elite ABC Standard Kit (Vector Laboratories).
LucTS reporter assay
pHSE-LucTS, pCRE-LucTS, and pSRE-LucTS were generated by replacing the Luc coding sequence of pHSE-Luc, pCRE-Luc, and
pSRE-Luc (all vectors from Clontech) with the LucTS. One day after transfection, MEFs were either treated with WTS, heat shock
(42 C), 100 mM forskolin, or 50% bovine serum. The mean values of the 4 h pre-stimulation baseline of lumi-nescence of pHSE-,
pCRE-, and pSRE-LucTS were set to 1. The Tet-inducible expression vector pTRE3G (Clontech) was used to construct the Per2
50 UTR-LucTS vectors. The 50 UTR region of the pTRE3G vector was substituted with the full-length Per2 50 UTR sequence of mouse
(NM_011066) or human (XM_006712824) origin. Mutagenesis of the Per2 m-uORF was performed using a standard sequential PCR
method.21 Tet-On 3G NIH3T3cells were transfected with a plasmid using the Viofectin reagent (Viogene). To reduce technical variation between transfections, the transfection was performed in a single dish and the cells were split into test dishes. D-luciferin
(Promega) was added to the medium before experiments.21 Cells were treated with doxycycline (1 mg/mL). Luminescence was
continuously monitored using a custom-modified dish-type luminometer AB-2550 Kronos Dio (ATTO), which enables the programming and recording of temperature profiles in real-time with an accuracy of 0.1 C. Luminescence was measured for 2 min at 30-min
intervals. Values were normalized to the luminescence intensity that was recorded 30 min before WTS. The fold increase was calculated by dividing the intensity of WTS-treated cells by that of non-treated controls.
Per2::LucTS cell-based kinase inhibitor assay
Per2::LucTS cells were treated with a compound in the Kinase Screening Library (Cayman Chemical, #10505) at a concentration of
10 mM/0.1% DMSO immediately before WTS. The score was calculated using the following formula;
Score = log2
Td of tested compound
Td of vehicle
where Td represents the difference in the half-maximum time between WTS and no-WTS cells. The results of compounds that profoundly affect basal LucTS activity were omitted. To evaluate dose dependency, Wortm was administrated at a concentration of 0.3,
1, 10, 30, 100, and 1000 nM. Where indicated, KNK437 (Sigma Aldrich, 100 mM), Rapamycin (Cayman chemical, 20 nM), MK2206
(Selleck chemicals, 10 mM), U0126 (Cayman chemical, 10 mM), DZnepA (Sigma Aldrich, 100 mM), D4476 (Cayman chemical,
5 mM), and Ruthenium Red (Wako, 10 mM) were used.
Quantification of PIP3 species
Cells were incubated in a serum-free medium 1 day before experiments and sampled afte ...
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