Volume 67, Issue 3, Pages 433-446.e4 (August 2017) Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals  Marco Preußner, Gesine Goldammer, Alexander Neumann, Tom Haltenhof, Pia Rautenstrauch, Michaela Müller-McNicoll, Florian Heyd  Molecular Cell  Volume 67, Issue 3, Pages 433-446.e4 (August 2017) DOI: 10.1016/j.molcel.2017.06.006 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Temperature Regulates U2af26 Alternative Splicing (A) U2af26 splicing after cold shock. N2A cells were shifted to 32°C (blue) for 2 hr, and U2af26 AS was analyzed by splicing-sensitive radioactive RT-PCR with primers in exons 4 and 8 (left, representative gel). Skipping of exon 6 (Δ6) and 6/7 (Δ67) was quantified and is depicted relative to 37°C (right). Data represent the mean of four independent experiments ±SD. ∗∗∗Student’s t test-derived p value < 0.001. (B) U2af26 splicing after heat shock. N2A cells were shifted to 42°C (red) for 2 hr, and AS was analyzed as in (A) and skipping of exons 6/7 was quantified relative to 37°C (n = 12, mean ± SD). ∗∗∗Student’s t test-derived p value < 0.001. (C) Temperature gradient. N2A cells were shifted to the indicated temperatures for 2 hr, and AS was analyzed and quantified as in (A). Data represent the mean of five to six independent experiments ± SD. The dotted line depicts a linear regression fit of temperature and exon skipping. (D) Square-wave temperature rhythm (top, temperature profile). N2A cells were harvested at the indicated hours (red box) and AS was analyzed, normalized to 72 hr (bottom, n ≥ 3, mean ± SD). Representative gel is on the right. (E) Shift to constant 37°C (top, temperature profile). After 2 days of square-wave rhythms as in (D), cells were transferred to 37°C, either from 38°C (red) or from 34°C (blue). Skipping of exons 6/7 was quantified and normalized to 48 hr (bottom, n ≥ 3, mean ± SD). (F) Correlation of U2af26 splicing and the circadian body temperature in mice. Post-mortem brain temperature in 6-week-old mice (left) and AS in cerebellum (middle) and liver (right) normalized to ZT7 (right, mean of n = 4 mice ± SD) are shown. ∗∗Student’s t test-derived p value < 0.01. See also Figure S1. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Temperature-Responsive Splicing in Mice (A) U2af26 splicing in young mice. Mice were kept under constant 12-hr light/dark cycles and analyzed at the indicated ZTs. Post-mortem brain temperature was measured in two mice (right y axis), and U2af26 AS was analyzed in cerebellum and liver (top: representative gel) of four mice as in Figure 1A, normalized to ZT0 (left y axis). Data represent the mean ± SD. ns, Student’s t test-derived p value > 0.05. (B) Effect of changes in environmental temperature. Mice of different ages (left: 21–42 days and right: 12–13 days) were kept at room temperature (RT) or at 18°C for 2 hr (cold). Post-mortem brain temperatures were measured (n ≥ 2), and AS was analyzed in cerebellum and liver of four to six mice. Mean ± SD. ∗∗∗Student’s t test-derived p value < 0.001. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 SRSF2 and SRSF7 Regulate U2af26 Alternative Splicing (A) Targeted siRNA screen. N2A cells were transfected with siRNA pools against the depicted SR proteins, and U2af26 AS was analyzed as in Figure 1A. The Δ67 isoform was quantified relative to ctrl siRNA (n = 2, mean ± SD, for untransfected n = 1). (B) Overexpression of SRSF2 and SRSF7. N2A cells were transfected with an empty vector or with overexpression vectors for SRSF2 or SRSF7, and AS was analyzed as in Figure 1A. On the right, western blot confirms overexpression of SRSF2 and SRSF7 (anti-FLAG), with HNRNPL as loading control. Mean of three independent experiments ±SD. ∗∗Student’s t test-derived p value < 0.01 and ∗p < 0.05. (C) iCLIP data of SRSF1–7 binding to the variable U2af26 region. The exon-intron structure of U2af26 is depicted at the bottom; the third quarter is highlighted in orange. CLIP tags in this region are shown for each SR protein. (D) Cold shock after SRSF2/7 knockdown. N2A cells as in (A) were shifted to 32°C (blue) and U2af26 AS was analyzed (top, representative gel). For each condition, the Δ67 isoform was quantified relative to 37°C (n = 4, mean ± SD). ∗∗∗Student’s t test-derived p value < 0.001. (E) Heat shock after SRSF2/7 knockdown. N2A cells as in (A) were shifted to 42°C (red) and AS was analyzed. For each condition, the Δ67 isoform was quantified relative to 37°C (n ≥ 3, mean ± SD). ∗∗∗Student’s t test-derived p value < 0.001 and ∗p < 0.05. (F) Square-wave temperature rhythm after SRSF2/7 knockdown. N2A cells were treated as in Figure 1D. After 24 hr, cells were transfected with the indicated siRNAs and harvested at the indicated times of the temperature rhythm. AS was analyzed and the Δ67 isoform was quantified relative to 72 hr for each condition (n = 3, mean ± SD). ∗∗Student’s t test-derived p value < 0.01 and ∗∗∗p < 0.001. See also Figure S2. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Temperature-Regulated SRSF2/7 Phosphorylation Controls U2af26 Alternative Splicing (A) Heat shock recovery. 3T3 cells were shifted to 42°C (red box) and returned to 37°C. U2af26 AS was analyzed and quantified relative to 37°C. Mean of three independent experiments ±SD. (B) Modulation of SR protein phosphorylation. 3T3 cells were treated with DMSO control, with OA (1 μM), or with TG-003 (50 μM) for 2.5 hr. U2af26 AS was analyzed (left, representative gel). Combined skipping of exons 6 and 7 and 5–7 was quantified relative to DMSO (right, n = 3, mean ± SD). ∗∗∗Student’s t test-derived p value < 0.001. See also Figures S3G and S3H. (C) Knockdown of Clk4. N2A cells were transfected with a pool of two independent siRNAs against Clk4 or a control siRNA, and U2af26 AS was analyzed as in Figure 1A. The Δ67 isoform was quantified relative to ctrl siRNA (left, n = 4, mean ± SD) ∗Student’s t test-derived p value < 0.05. On the right, qPCR confirms knockdown of Clk4. Expression is depicted relative to Hprt and ctrl siRNA (right, n = 3, mean ± SD). ∗Student’s t test-derived p value < 0.05. (D) Temperature-dependent SR protein phosphorylation. P19 cells were shifted to 32°C or 42°C, and SR protein phosphorylation was analyzed by western blotting with an antibody that specifically recognizes phosphorylated SR residues (α1H4, the size of SRSF2/7 is marked by an arrowhead). On the left, size marker in kDA is shown. SR protein phosphorylation (total α1H4 signal) was quantified relative to GAPDH (n ≥ 3, mean ± SD). ∗∗Student’s t test-derived p value < 0.01 and ∗p < 0.05. (E) SRSF2 and SRSF7 phosphorylation. P19 cells stably expressing GFP-tagged SRSF2 (top) or SRSF7 (bottom) were shifted to 32°C or 42°C, and nuclear extracts were analyzed by western blotting against GFP. The size of phosphorylated (red P) or dephosphorylated proteins is labeled on the right. HNRNPL served as a loading control. (F and G) Phosphorylation-dependent IPs. P19 cells stably expressing GFP-tagged SRSF2 (F) or SRSF7 (G) were shifted to 32°C, and lysates were immunoprecipitated with the α1H4 antibody, including a P19 cell line stably expressing GFP as a control and a control without antibody (-AB). Input and IP samples were analyzed by western blotting against GFP (hc, heavy chain). GAPDH served as a loading control. GFP signals in IPs were quantified relative to the input signal and 37°C (mean of three to four independent experiments ±SD). ∗Student’s t test-derived p value < 0.05. See also Figure S3. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Temperature-Regulated SR Protein Phosphorylation in Mice (A) Temperature-regulated SR protein phosphorylation in mouse. Liver lysates from 12-day-old mice as in Figure 2B were analyzed by western blotting with the α1H4 antibody (the size of SRSF2/7 is marked by an arrowhead). SR protein phosphorylation was quantified relative to GAPDH and RT (right, mean of three independent mice ± SD). ∗Student’s t test-derived p value < 0.05. (B) Liver nuclear extracts from 6-week-old mice at different ZTs were analyzed as in (A) and quantified relative to HNRNPL and ZT8 (right, mean of three independent mice ±SD) ∗∗Student’s t test-derived p value < 0.01. (C) Liver (left) and cerebellum (right) nuclear extracts from ZT0 and ZT8 were immunoprecipitated with the α1H4 antibody and analyzed by western blotting against SRSF7. HNRNPL served as a loading control. SRSF7 signal was compared in IP and input (mean of three to four independent mice ±SD). ∗Student’s t test-derived p value < 0.05 and ∗∗∗p < 0.001. See also Figure S4. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 A Splicing-Based Thermometer Globally Controls Oscillating Alternative Splicing (A) Identification of cold-responsive splicing events by RNA-seq. Identified events are categorized into alternative 3′ ss (A3′ss), alternative 5′ ss (A5′ss), mutually exclusive exons (Mxes), retained intron (RI), and skipped exon (SE). U2af26 exon 6 was identified as a skipped exon event. RNA-seq data from Liu et al. (2013). (B) Square-wave temperature rhythm. In N2A cells as in Figure 1D, AS of four exemplary targets was analyzed by splicing-sensitive radioactive RT-PCR and splicing isoforms were quantified. For each target, percentage of one isoform (for SE events always the skipping isoform) was calculated and depicted relative to 72 hr (n = 3, mean ± SD). (C) Sensitivity of temperature-responsive splicing. N2A cells were incubated for 12 hr at 36°C, 36.5°C, 37.5°C, or 38°C, and AS of 17 targets was analyzed as in (B) and calculated relative to 36°C. Data represent the mean of three independent experiments ±SD (for 36°C n = 2). Student’s t test-derived p values were calculated relative to 38°C (ns, p > 0.05; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). up, upstream; dn, downstream. See also Table S2. (D) Rhythmic splicing in mouse cerebellum. RNAs from the indicated ZTs were analyzed as in (B). Representative gels are on top (the asterisks mark unspecific PCR products) and quantifications are below (mean of at least three mice ±SD). Student’s t test-derived p values ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S5. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 A Thermometer-like Splicing Switch in the Tbp 5′ UTR Generates Oscillating TBP Protein Expression (A) Temperature gradient response of Tbp AS. Top: exon-intron structure is shown, with skipping of exons X and Y in red (ΔXY). Bottom: splicing was analyzed by radioactive RT-PCR and the percentage of the ΔXY isoform is shown relative to 35°C. The dotted line depicts a linear regression fit of temperature and the ΔXY isoform (n = 3, mean ± SD). (B) Square-wave temperature rhythm. Splicing was analyzed as in (A) and is depicted relative to 72 hr (n = 3, mean ± SD). (C) Tbp 5′ UTR-dependent luciferase expression. Alternative UTRs were cloned in front of a firefly luciferase reporter (left). After transfection in N2A cells, firefly activity was analyzed relative to a renilla control and normalized to the ΔY reporter (right, n ≥ 6, mean ± SD). ∗∗∗Student’s t test-derived p value < 0.001. Bottom: predicted secondary structures formed by the variable Tbp region are shown. (D) Temperature-regulated TBP expression. N2A cells were kept for 4 hr at the indicated temperatures and analyzed for TBP expression using western blotting (left). TBP expression is shown relative to HNRNPL and 39°C (right, n = 6, mean ± SD). ∗∗Student’s t test-derived p value < 0.01. (E) Oscillating Tbp splicing in mouse cerebellum. Splicing was analyzed as in (A) (mean of at least three mice ±SD). Student’s t test-derived p values ∗p < 0.05 and ∗∗p < 0.01. (F) Oscillating TBP expression in cerebellum. Lysates from the indicated ZTs were analyzed by western blotting as in (D) (mean of three (two for ZT20) independent mice ±SD). ∗Student’s t test-derived p value < 0.05. (G) TATA-box-dependent oscillation in gene expression. Genes with cycling transcription (Amplitude Nascent Seq > 0.1) in liver were extracted from Westermark (2016) and separated into TATA-box-containing (n = 458) and -non-containing genes (n = 3,835). The peak in expression (ZT RNA-seq) was compared between the two groups (Mann-Whitney test-derived p value ∗∗∗p = 0.0002). (H) Model for AS regulation through body temperature cycles resulting in maximal TBP expression and, consequently, highest transcription rate at the beginning of the night. See also Figure S6. Molecular Cell 2017 67, 433-446.e4DOI: (10.1016/j.molcel.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions