1. Volume 54, Issue 4, Pages 651-662 (May 2014)
Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice 
Marco Preußner, Ilka Wilhelmi, Astrid-Solveig Schultz, Florian Finkernagel, Monika Michel, Tarik Möröy, Florian Heyd 
Molecular Cell 
Volume 54, Issue 4, Pages (May 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 54, 651-662DOI: (10.1016/j.molcel.2014.04.015)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Circadian Alternative Splicing of U2af26 Exons 6 and 7 in Peripheral Clocks
(A) Rhythmic U2af26 alternative splicing in mouse cerebellum. Mice were kept under constant 12 hr LD conditions (white bar, light on; black bar, light off), sacrificed at the indicated zeitgeber times (ZT), and splicing sensitive radioactive RT-PCR was performed using cerebellum RNA and primers to U2af26 exons 4 and 8. HPRT served as a loading control. Schematic representation of U2af26 splicing isoforms on the right: U2AF26fl containing exons 4–8 and U2AF26ΔE67 lacking exons 6 and 7. On the right, quantification of percent ΔE67 and U2AF26fl/HPRT is shown from two independent time courses with at least three mice per time point (for U2AF26fl ZT20, n = 2).
(B) Entrained U2af26 alternative splicing. Mice were 8 hr phase delayed and on the fourth day sacrificed at the indicated ZTs. RT-PCR analysis as in (A) and quantification of %ΔE67 in red (n = 3). The skipping ratio before entrainment is shown in light gray.
(C) U2af26 alternative splicing persists in the absence of external cues. Mice were kept in constant darkness for 24 hr and sacrificed at the indicated circadian times of the following subjective day. Dark gray bars represent subjective day with light off. RT-PCR analysis, and quantification of %ΔE67 was performed as in (A) (n = 4; p < ).
(D) U2af26 alternative splicing is not regulated in the SCN. RT-PCR using SCN RNA from the indicated ZTs was performed as in (A). Since the SCN clock is 4 hr advanced, we chose ZTs 4 and 12, corresponding to ZTs 8 and 16 in peripheral clocks. On the right quantification of full length (% E4–8) is shown (n = 3).
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Skipping of U2af26 Exons 6 and 7 Generates a Domain with Homology to Drosophila TIM
(A) Scheme illustrating the exon-skipping (U2AF26ΔE67) induced frameshift resulting in translation far into the 3′ UTR, thereby generating 143 new amino acids.
(B) BLAST search with the novel C-terminal domain reveals a homology to the Drosophila TIM protein (TIM homology domain, THD). The alignment reveals 28% identical (∗), 48% positive (:), and 67% tolerated (.) amino acids; shown is the alignment of the last 100 amino acids.
(C) Circadian expression of the THD domain. Mice were kept and sacrificed as in Figure 1A; cerebellum lysates were prepared at the indicated ZTs and analyzed by western blot using an antibody against the THD. GAPDH served as a loading control. Quantification is shown below (n = 2).
(D and E) Cytoplasmic localization of U2AF26ΔE67. In (D), liver proteins from mice sacrificed at ZT8 were separated in cytoplasmic and nuclear extracts (CTX/NX) and analyzed by western blot with an antibody against the THD. In (E) HeLa cells were transfected with GFP-U2AF26ΔE67 or GFP alone, and CTX and NX were separated and analyzed by western blot with an antibody against GFP. GAPDH served as a CTX marker and hnRNP L as a marker for NX.
(F) U2AF26 is destabilized by the THD domain. Hek293 cells were transfected either with U2AF26fl-GFP or with GFP-U2AF26ΔE67 and incubated with Cycloheximide (CHX) for the indicated times before harvest. Western blot with a GFP antibody was performed to monitor U2AF26 levels. GAPDH served as a loading control.
(G) Proteasomal degradation of U2AF26ΔE67. GFP-U2AF26ΔE67-transfected Hek293 cells were treated with CHX and with or without the proteasome inhibitor MG132 for the indicated times before harvest. Western blot was performed as in (F).
(H) Quantification of experiments as shown in (F) and (G) (n > 3; ∗p < 0.05; ∗∗∗p < 0.001).
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 PER1 Interacts with U2AF26 via Its PasB Domain
(A) Specific coprecipitation of U2AF26ΔE67 with PER1-Flag. Hek293 cells were cotransfected with GFP (right) or GFP-U2AF26ΔE67 (left) and either empty vector or PER1-Flag. Immunoprecipitations were performed using M2-Flag agarose. Coimmunoprecipitations were analyzed by western blot with anti-Flag and anti-GFP antibodies. Representative blots from three experiments are shown (h.c., heavy chain; l.c., light chain).
(B) Immunoprecipitations were performed as in (A), but with the indicated amounts of NaCl and TritonX. Complex formation of PER1-Flag and GFP-U2AF26ΔE67 is stable up to 1.2M NaCl and 2% TritonX.
(C) PER1 coprecipitates with U2AF26ΔE67. Hek293 cells were cotransfected with MS2-Flag-U2AF26ΔE67 and PER1. Immunoprecipitations were performed as in (A).
(D) Endogenous U2AF26ΔE67 and PER1 coprecipitate. Cytoplasmic extract (CTX) from WT or U2AF26-deficient (KO) livers, harvested at ZT8, was used in IPs with beads alone (ctrl.) or an antibody against the THD (aTHD). Input and precipitates were analyzed with the indicated antibodies by western blot.
(E) In contrast to U2AF26fl (26), the THD does not coprecipitate with PER1. Cells were cotransfected with PER1-Flag and either U2AF26fl-GFP (26) or GFP-THD (THD). Immunoprecipitations and western blot as in (A).
(F) Mapping of the PER1 interaction domain within U2AF26 to the first zinc finger (ZNF1). Neither the RNA recognition motif (RNP 1 and 2) nor the second zinc finger (ZNF2) was required for coprecipitation (see also Figure S3A).
(G) U2AF35 coprecipitates with PER1. Cells were cotransfected with U2AF35-GFP and either empty vector or PER1-Flag. Immunoprecipitations were performed as in (A).
(H) Mapping of the U2AF26 interaction domain within PER1 to the PasB domain. On the right, exemplary coimmunoprecipitations are shown for the interacting PasB domain and the none-interacting C terminus (ΔCk1). See also Figure S3B.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 U2AF26ΔE67 Promotes Proteasomal Degradation of PER1
(A) PER1 half-life is specifically decreased by U2AF26ΔE cells were cotransfected with PER1-Flag and either GFP or the indicated U2AF26 variants, and treated with CHX for the indicated times. Western blot with a Flag antibody was performed to monitor PER1 levels. GAPDH serves as a loading control. Quantification is shown on the right (n > 3; ∗p < 0.05).
(B) U2AF26ΔE67 selectively destabilizes the interacting PasB domain. PER1 deletion constructs (PasB or ΔCk1) were cotransfected either with GFP or with U2AF26ΔE67. CHX-treatment and western blots were performed as in (A). GAPDH or hnRNP L served as loading control. Below quantifications are shown (n = 3; ∗p < 0.05; ∗∗p < 0.01).
(C) Proteasomal PER1 degradation. GFP-U2AF26ΔE67/Per1-Flag-cotransfected 293 cells were treated with CHX and with proteasome inhibitors MG132 (MG) or Bortezomib (Borte) for the indicated times before harvest. Western blot was performed as in (A). Below quantification is shown; Bortezomib prevents U2AF26ΔE67-induced PER1 degradation (n = 3; ∗p < 0.05).
(D) PER1 is stabilized in U2AF26ko liver. Liver cells were isolated from WT or KO mice sacrificed at ZT8 and cultured in the presence of CHX for the indicated times. Western blot was performed to monitor PER1 levels using GAPDH as a loading control. Quantification of PER1 stability is shown on the right (n > 4; ∗p < 0.05).
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
U2AF26-Deficient Animals Show Defects in Circadian Gene Expression
(A) PER1 protein oscillation in liver of wild-type (WT) and U2AF26-deficient (KO) animals. Western blotting was performed using total liver lysates with the indicated antibodies.
(B) Quantification confirms acyclic PER1 expression in KO animals (n = 3 for each time point, two independent time courses; ∗p < 0.05).
(C) Western blot from liver cytoplasmic extracts confirming induced expression of U2AF26ΔE67-protein at ZT4 and 8.
(D and E) RT-qPCR analysis of clock-gene mRNA expression in liver (D) or cerebellum (E) of WT and KO animals. Significant differences comparing WT and KO animals were observed for all analyzed genes (n > 3 from two independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Altered Clock Resetting in U2AF26-Deficient Animals
(A) Actograms of two representative WT and KO animals in response to a 4 hr phase advance. The arrows indicate faster adaptation on the first day of entrainment in KO animals.
(B) Quantification of the activity onset over 4 days. U2AF26-deficient animals show a significantly faster re-entrainment on the first day (∗∗∗p < 0.001).
(C) Fast entrainment of U2af26 splicing in cerebellum. Mice were 8 hr phase advanced and sacrificed at the indicated ZTs. RT-PCR analysis was performed as in Figure 1A; quantification of %ΔE67 in cerebellum and liver is shown in red (n > 3). The skipping ratio before entrainment is shown in light gray for cerebellum.
(D) Altered Per1 mRNA expression in cerebellum of phase-advanced KO animals. RT-qPCR analysis of mice phase advanced as in (C) from ZT8, comparing cerebellum and liver from WT and KO animals (n > 3; ∗∗p < 0.01; n.s., not significant).
(E) Model comparing the roles of TIM and U2AF26ΔE67 in light-induced control of PER1 protein and clock resetting.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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