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Cold-induced RNA-binding proteins regulate circadian gene expression by controlling alternative polyadenylation

The body temperature is considered a universal cue by which the master clock synchronizes the peripheral clocks in mammals, but the mechanism is not fully understood. Here we identified two cold-induced RNA-binding proteins (RBPs), Cirbp and Rbm3, as important regulators for the temperature entrained circadian gene expression. The depletion of Cirbp or Rbm3 significantly reduced the amplitudes of core circadian genes. PAR-CLIP analyses showed that the 3′UTR binding sites of Cirbp and Rbm3 were significantly enriched near the polyadenylation sites (PASs). Furthermore, the depletion of Cirbp or Rbm3 shortened 3′UTR, whereas low temperature (upregulating Cirbp and Rbm3) lengthened 3′UTR. Remarkably, we found that they repressed the usage of proximal PASs by binding to the common 3′UTR, and many cases of proximal/distal PAS selection regulated by them showed strong circadian oscillations. Our results suggested that Cirbp and Rbm3 regulated the circadian gene expression by controlling alternative polyadenylation (APA).


IV. Supplementary Tables
Supplementary Table S1 -Reads and clusters generated by PAR-CLIP.
Supplementary Table S2 -Cirbp-and Rbm3-binding sites that located in the gene regions. Supplementary

Retrovirus plasmids and infection
For the over-expression of Flag-tagged Cirbp/Rbm3 in the MEFs, the mouse Cirbp and Rbm3 cDNAs were amplified by PCR (The KOD Hot Start DNA Polymerase kit, TOYOBO) from mouse cDNA library and cloned into the p3xFLAG-CMV-5' vectors, then the cDNAs of the FLAG-tagged Cirbp/Rbm3 were cloned into pMSCV/Pro vectors (Clontech). For knockdown of Cirbp and Rbm3 in MEFs, retrovirus-mediated siRNA expression was used. The target sequences were determined using the Whitehead Institute siRNA designing tool (Yuan et al., 2004) and verified by BLAST searches to ensure specificity. As recommended by Clontech for the RNAi-Ready pSIREN-RetroQ were plated into 6-well plates before retroviral infection. Virus-containing supernatants were supplemented with 20 µg/ml polybrene and 20 µg/ml HEPES, and then added to the cells for a spin infection procedure, which was performed in the 6-well plates by centrifugation at 2500 rpm at 30°C for 1.5 h. The mock cells were performed with all the procedures, and were treated with the normal DMEM medium rather than the virus containing supernatants in the retroviral infection step. The MEFs were selected with 50 µg/ml puromycin.

RNA extraction and qPCR
Total RNA was isolated from cells and tissues by Trizol (Invitrogen). Total RNA quantities were measured by a Nanodrop spectrometer. RNA quality was assessed by an Agilent Bioanalyzer. 500 ng of total RNA was reverse transcribed using random hexamer and the Superscript II reverse transcriptase (Invitrogen). 5 μl of RT product (1:10 diluted) and SYBR Green I Master Mix (Roche) were used in qPCR on a LightCycler 480 (Roche). The specificity of PCR was checked by melting curve analysis. In every qPCR assay, Actb or Gapdh was used as the control for the significant bias of starting materials across samples. Primers for qPCR analyses are listed in Table S10.

Western blot and antibodies
Liver samples of mice were washed with ice-cold PBS three times to remove blood and then homogenized in lysis buffer (8M urea, 4% CHAPS, 65mM DTT, 40mM Tris with protease inhibitor mixture, 200 mg of wet tissue/ml) with a motor-driven homogenizer (Glas-Col) until no sample pieces were visible. The homogenate was sonicated for 3 min followed by centrifugation at 15,000 g for 1 h at 4°C, and the supernatant was separated by SDS-PAGE. The proteins were transferred to PVDF membrane using wet blotting system (BioRad). The membrane was blocked in 5% non-fat milk and incubated with primary antibody for 1 h to overnight, washed with TBST twice and incubated with secondary antibody for 1 h. The protein bands were visualized using ECL reagents (GE Healthcare) and LAS-4000 CCD camera (FUJIFILM). All bands were quantified using Multi Gauge Software (FUJIFILM). Primary antibodies used were the antibody to Cirbp (LifeSpan BioSciences), dilution 1:500; the antibody to Rbm3 was a gift from Dr. Peter W. Vanderklish, dilution 1:500; the antibodies to Actb and Gapdh were from Dr. Gang Wang, dilution 1:1000. The secondary antibody was HRP-conjugated anti-mouse IgG (Abcam), dilution 1:10000.

Tandem UTRs database construction
Mm9 KnownGene annotations were extracted from UCSC -f -t --chunkmbs 1024 --best -n 0 -p 6 -k 20 -m 20 mm9). The mapping results were filtered so that the mapping is perfect (no mismatch) and unique on 3' UTR. We selected the sub-UTRs with at least 3 mapped probes. The probes were then grouped into probesets. Finally, we obtained 6220 tandem UTR events in 4364 genes, and generated a CDF annotation for Affymetrix Mouse Genome 430 2.0 platform.

RNA-seq
Polyadenylated RNA from the mock cells at 37°C or 32°C with two replicates, siCirbp-1, siCirbp-2, siRbm3-1 and siRbm3-2 MEFs at 37°C were sequenced on Illumina GAII using 76 bp single-end kits according to the manufacturer's instructions. The detailed information was shown in conditions. For example, to identify the significantly changed PAS-usage switching events upon Cirbp depletion, the 2 x 2 table was constructed from the read counts in the common 3'UTRs in the mock cells, the read counts in the extended 3'UTRs in the mock cells, the read counts in the common 3'UTRs in the Cirbp-depleted cells and the read counts in the extended 3'UTRs in the Cirbp-depleted cells.

Microarray Analysis
Microarray raw data were downloaded from ArrayExpress repository (E-MEXP-842) and GEO (GSE11923). The data were extracted using RMA implemented in R. Present/absent calls were generated by MAS5 in R for Mouse Genome 430 2.0 expression arrays. The annotations including gene names, gene symbols, RefSeq accessions were obtained from R Bioconductor package (mouse4302.db). The COSOPT(Straume, 2004) algorithm was used to measure the goodness-of-fit between experimental data and cosine curves with varying phases and different period lengths.
P-values were then calculated by fitting it to cosine curves in order to determine the probability that the observed data matches a cosine curve by chance alone. Pearson correlation was used to estimate the correlation of the expression level between liver clock active and arrested mice. For identifying the genes that showed oscillating ext/com UTR-ratio, the expression level of the sub-UTR was obtained from the microarray data of the mouse liver (GSE11923). Then we calculated the ext/com UTR-ratio in each time point, and estimated the statistical significance of the oscillation by the COSOPT algorithm.

RIP and qPCR
The cells that are stably expressing the FLAG epitope-tagged protein of interest were harvested, washed in ice-cold PBS and collected by centrifugation (1500g, 4°C and 10 min

PAR-CLIP procedure
PAR-CLIP protocol was performed as described (Hafner et al., 2010). The cells that are stably expressing the FLAG epitope-tagged protein of interest were grown in medium supplemented with 100µM 4SU for 16h prior to harvest.

UV 365 nm crosslinking
For UV-crosslinking, the growth medium was removed completely while the cells were still

Cell lysis and first partial RNase T1 digestion
The pellets of the cells crosslinked with UV 365 nm were resuspended in 3 cell pellet volumes of NP40 lysis buffer (50 mM Tris HCl, pH 7.5, 140 mM LiCl, 2 mM EDTA, pH 8.0, 0.5% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail (Roche)) and incubated on ice for 10 min. The typical scale of such an experiment was 3 ml of cell pellet. The cell lysate was cleared by centrifugation at 13,000g. RNase T1 (Fermentas) was added to the cleared cell lysates to a final concentration of 1 U/μl and the reaction mixture was incubated in a water bath at 22°C for 10 min and subsequently cooled for 5 min on ice before the addition of antibody-conjugated magnetic beads.

Preparation of Dynabeads Protein G magnetic beads
10 μl of Dynabeads Protein G magnetic particles (Invitrogen) per ml cell lysate were washed twice with 1 ml of citrate-phosphate buffer (4.7 g/l citric acid, 9.2 g/l Na2HPO4, pH 5.0) and resuspended in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.
0.25 μg of anti-Flag M2 monoclonal antibody (Sigma) per ml suspension was added and incubated at room temperature for 40 min. Beads were then washed twice with 1 ml of citrate-phosphate buffer to remove unbound antibody and resuspended again in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.

Immunoprecipitation, second RNase T1 digestion and dephosphorylation
10 μl antibody-conjugated Protein G magnetic beads were added per ml of partial RNase T1 treated cell lysate. Incubation was performed in 1.5 ml microfuge tubes on a rotating wheel for 1 h at 4°C.
Magnetic beads were collected on a magnetic particle collector (Invitrogen). Manipulations of the following steps were carried out in 1.5 ml microfuge tubes. The supernatant was removed from the bead-bound material. The beads were washed 2 times with 1 ml of IP wash buffer (

Radiolabeling of RNA segments crosslinked to immunoprecipitated proteins
To the bead suspension described above, γ-32 P-ATP was added to a final concentration of 0.25 μCi/μl and T4 Polynucleotide Kinase (PNK) to 1 U/μl in one original bead volume. The suspension was incubated for 30 min at 37°C. Thereafter, nonradioactive ATP was added to obtain a final concentration of 100 μM and the incubation was continued for another 5 min at 37°C. The magnetic beads were then washed 5 times with 800 μl of PNK Buffer and resuspended in 20 μl of SDS-PAGE Loading Buffer (10% glycerol (v/v), 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 2% SDS (w/v), 100 mM DTT, 0.1% bromophenol blue).

SDS-PAGE and Western Blotting
Immunoprecipitation of epitope-tagged protein of interest was performed as described in the above section and radioactively labeled at the 5'end. Following radiolabeling, the beads were washed 5 times with 800 μl of PNK Buffer. The beads were resuspended in 50 μl of SDS-PAGE loading buffer and boiled 5 min at 95°C. The supernatant was loaded on a 4-12 % NuPAGE Bis-Tris gradient gel (Invitrogen) and exposed to a phosphorimager screen for the detection of radioactive signal. The gel was subsequently transferred to a nirocellulose membrane and protein-RNA complexes were immunodetected by incubation with Flag specific antibody, and the tagged protein was visualized using ECL Western Blot detection reagent (GE-Healthcare).

Electroelution of crosslinked RNA-protein complexes from gel slices
The radioactively-labeled RNA-protein complex migrating at the expected molecular weight of the target protein was excised from the gel and electroeluted in 800 μl SDS running buffer using D-Tube Dialyzer Midi (Novagen) according to the instructions of the manufacturer.

Proteinase K digestion
An equal volume of 2x Proteinase K Buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 12.5 mM EDTA, 2% (w/v) SDS) with respect to the electroeluate was added, followed by the addition of Proteinase K (Roche) to a final concentration of 1.2 mg/ml, and incubation for 30 min at 55°C. The RNA was recovered by acidic phenol/chloroform extraction followed by chloroform extraction and ethanol precipitation. The pellet was dissolved in 6 μl water.

cDNA library preparation and deep sequencing
The recovered RNA was carried through a cDNA library preparation protocol, originally described for cloning of small RNAs (Dolken et al., 2008;Hafner et al., 2008). In contrast to the published cloning procedure, the 3'adapter ligation was carried out with a barcoded 3'adapter in a 20 μl reaction volume using 6 μl of the recovered RNA. PAR-CLIP libraries were sequenced on Illumina Genome Analyzer GAII platform using the 1X50BP single read protocol.

Generation of binding clusters and crosslinking positions
All PAR-CLIP sequencing data were analyzed with a computational analysis pipeline (Lebedeva et al., 2011). Illumina PAR-CLIP cDNA sequencing reads were aligned to the mouse genome assembly (mm9) allowing for up to one mismatch, insertion or deletion. Only uniquely mapped reads were retained. We identified the clusters of aligned reads continuously covering genomic regions based on the condition that a cluster requires 20 and more overlapping reads and at least one T-to-C transition event. The number of T-to-C mismatches served as a crosslink score, and the position with the highest frequency of T-to-C transition was referred to as the preferred crosslinking position. As the reads should originate from protein-bound transcripts, we regarded clusters aligning antisense to the annotated direction of transcription as false positives.

Conservation profiles analysis
Genome-wide conservation profiles per nucleotide PhyloP scores (Pollard et al., 2010) were obtained via UCSC table browser. The PhyloP scores in a 201nt window around the preferred crosslink site of each binding site were obtained and averaged over specific category of binding clusters. The result was a high resolution profile of average nucleotide conservation scores at a given distance from the preferred crosslink sites. The randomly selected region within the same transcript was used as a measure of background conservation.

III. Supplementary Figures
Supplementary Fig. S1. The RBPs and HSPs that showed highly similar circadian expression patterns in liver clock active and arrested mice. Related to Table 1

IV. Supplementary Tables
Supplementary Table S1. Reads and clusters generated by PAR-CLIP.