Rhythmic U2af26 alternative splicing controls PERIOD1 stability and the circadian clock in mice.

Please cite this article in press as: Preußner et al., Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.04.015

Molecular Cell

Article Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice Marco Preußner,1,3 Ilka Wilhelmi,1,3 Astrid-Solveig Schultz,1,3 Florian Finkernagel,1 Monika Michel,1 Tarik Mo¨roÿ,2 and Florian Heyd1,3,* 1Philipps-University Marburg, Institute of Molecular Biology and Tumor Research (IMT), Emil-Mannkopff-Strasse 2, 35032 Marburg, Germany 2Institut

de Recherches Cliniques de Montreál (IRCM), 110 Avenue des Pins Ouest, Montreál, QC H2W 1R7, Canada address: Free University Berlin, Institute of Chemistry and Biochemistry, Takustrasse 6, 14195 Berlin, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.04.015 3Present

SUMMARY

The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. Here we describe a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2auxiliary-factor 26 (U2AF26). This results in translation far into the 30 UTR, generating a C terminus with homology to the Drosophila clock regulator TIMELESS. This new U2AF26 variant destabilizes PERIOD1 protein, and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest lightinduced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions.

INTRODUCTION The circadian clock creates a cell-autonomous rhythm with a period of approximately 24 hr that controls expression of diverse target genes through interlocking transcriptional/translational feedback loops (Dibner et al., 2010). The presence of a circadian clock in all domains of life and its connection to several diseases in mammals underline its fundamental importance for cellular homeostasis (Takahashi et al., 2008). In mammals the central clock, residing in the suprachiasmatic nucleus (SCN), uses external cues such as the light:dark (LD) cycle to synchronize peripheral clocks. Entrainment of the circadian clock to changed LD conditions, i.e., upon experimental jet lag, is slow in mammalian systems, indicating the presence of a buffering system to

reduce the effect of sudden light changes. Even though this is a long-known feature of the clock, its molecular details have only recently begun to emerge (Jagannath et al., 2013; see Discussion), and it remains unknown if such a system exists in peripheral clocks. At the core of the clockwork heterodimers of the basic helixloop-helix/Pas-containing transcription factors, CLOCK and BMAL1 drive rhythmic expression of clock controlled genes, among them Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes (Gekakis et al., 1998; Bunger et al., 2000). Accumulating PER proteins form complexes with CRY proteins in the cytoplasm, resulting in their translocation to the nucleus, where they repress the CLOCK/BMAL1 complex and thereby their own transcription (Lee et al., 2001). In Drosophila TIMELESS (TIM) substitutes CRY as the PER heterodimerization partner; a homolog of Drosophila TIM exists in mammals, but its function is less well defined (Gotter, 2006). Several lines of evidence have recently emphasized the impact of posttranscriptional regulation for the circadian rhythm. One example is the rather small percentage of circadian genes that are regulated by de novo transcription (Reddy et al., 2006; Menet et al., 2012; Koike et al., 2012); furthermore, control of the stability of core clock proteins such as CRY1 and CRY2 by ubiquitination has emerged as a central mechanism to regulate the circadian clock in mammals (Yoo et al., 2013; Hirano et al., 2013). Similarly, RNAbinding proteins such as LARK, ATAXIN-2, hnRNP Q, CIRP, or 4E-Bp1 regulate the expression of core clock components at the level of mRNA localization, stability, and translation (Kojima et al., 2007; Lee et al., 2012; Morf et al., 2012; Lim and Allada, 2013; Zhang et al., 2013; Cao et al., 2013). Interestingly, the described regulatory mechanisms control expression of clock components either at the transcriptional or the mRNA level (i.e., pre- or postsplicing), with a potential role of alternative splicing in regulating the mammalian circadian clock remaining enigmatic. Alternative splicing is recognized as a mechanism that multiplies the genome’s coding capacity and has a vast, yet largely unexplored, regulatory potential (Irimia and Blencowe, 2012). Misregulation of alternative splicing has been connected to many human diseases, neurological disorders and cancer, among others (Cooper et al., 2009; David and Manley, 2010), underlining its crucial regulatory function. Furthermore, recent Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 1


Molecular Cell Circadian U2af26 Alternative Splicing

studies have identified examples of signal-induced alternative splicing that control protein expression and function in response to external stimuli independently of regulated transcription (Lee et al., 2009; Heyd and Lynch, 2010). However, in contrast to recent discoveries in plants (Sanchez et al., 2010), a connection between alternative splicing and the circadian clock is only beginning to be appreciated in mammalian systems. Even though some circadian alternative splicing events have been described in mice, their functional significance remains unknown (McGlincy et al., 2012). U2AF26 is one of several homologs of the small U2 auxiliary factor (U2AF) subunit U2AF35. U2AF homologs are expressed in a tissue-specific manner (Mollet et al., 2006), but their functional role remains mostly unknown. U2AF26 can substitute U2AF35 in in vitro splicing reactions (Shepard et al., 2002) but has been suggested to fulfill a nonredundant function in Cd45 alternative splicing in vivo (Heyd et al., 2006). In mice, relative expression of both genes is regulated in a tissue-specific manner with high expression of U2af26 mRNA in brain (Shepard et al., 2002; also see Figure S1A online). U2af26 itself is alternatively spliced, giving rise to at least three mRNA isoforms (Heyd et al., 2008; also see Figure S1B). These findings and the initial identification of U2af26 in a screen for genes potentially regulated by the circadian transcription factor NPAS2 (Shepard et al., 2002) prompted us to investigate U2af26 alternative splicing with the circadian clock. Here we provide evidence for circadian and light-inducible U2af26 alternative splicing in mouse brain and liver that, by introducing a frameshift, generates a protein domain with homology to the Drosophila clock regulator TIM. In analogy to TIM, this U2AF26 variant regulates expression of the central clock component PER1, leading to a broad influence on circadian gene expression. Furthermore, our data suggest light-induced U2af26 alternative splicing to limit re-entrainment following experimental jet lag, providing a molecular mechanism that contributes to the delayed adaptation of the mammalian circadian clock. RESULTS Circadian Alternative Splicing of U2af26 Exons 6 and 7 Given the high expression of U2af26 in mouse brain (Shepard et al., 2002; Figure S1A) and the existence of at least three different U2af26 isoforms (Heyd et al., 2008; Figure S1B), we were interested in further characterizing U2af26 expression in brain. A connection between U2AF26 and the circadian transcription factor NPAS2 has been suggested (Shepard et al., 2002), which prompted us to focus on U2af26 isoform expression with the circadian clock. To this end, we first used cerebellum as a brain region with well-established circadian oscillation in gene expression. Wild-type (WT) mice were kept under 12 hr LD conditions, and RNA from cerebellum was prepared every 4 hr. Using splicing-sensitive RT-PCR we detected mainly two U2af26 isoforms: full-length (fl) and the one resulting from skipping of exons 6 and 7 (DE67). The DE67 isoform changed approximately 5-fold across the day: from 7% at zeitgeber time 0 (ZT0: light-on time point) to over 30%, with a peak at ZT8 (Figure 1A, zeitgeber is an external cue, here light, that synchronizes/entrains biological rhythms). We observed a 2 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

concomitant reduction in the fl isoform resulting in total U2af26 remaining constant (Figure S1C), and RT-qPCR confirmed that U2af26 is not substantially regulated at the transcriptional level (Figure S1D). Consistently, the neighboring gene Psenen, which has been suggested to share a bidirectional promoter with U2af26 (Didych et al., 2013), does not show rhythmic mRNA expression (Figure S1E). The observed change in isoform expression was specific for U2af26, as a corresponding switch in isoform expression was not detected in the close homolog U2af35, where only the full-length isoform was detected (Figure S1F). When we investigated U2af26 isoform expression over 2 consecutive days, we observed U2af26 exons 6 and 7 skipping rates to be identical at the corresponding ZTs, suggesting a period of 24 hr (Figure S1G). To confirm that the change in U2af26 isoform expression was mediated by a splicing switch, as opposed to regulated mRNA stability, we used MEFs that upon Dexamethasone shock (a standard method to synchronize the circadian clock of cells in culture) partially recapitulate rhythmic U2af26 isoform expression (Figure S1H). After a 4 hr block of de novo transcription using Actinomycin D, we compared the abundance of U2AF26fl and DE67 mRNAs at time points showing minimal and maximal exon exclusion, respectively. As the U2AF26 fl:DE67 ratio was not significantly altered by Actinomycin D under either condition (Figure S1I), we conclude that mRNA stability is not substantially regulated, suggesting that an alternative splicing switch accounts for rhythmic U2af26 isoform expression. To confirm a circadian splicing switch, we tested its persistence in dark:dark (DD) conditions and its entrainability by shifted LD cycles (Figures 1B and 1C). Phase-delaying mice by 8 hr (i.e., prolonging one night by 8 hr and then continuing a 12 hr LD cycle) also resulted in an 8 hr phase shift in U2af26 alternative splicing. Furthermore, by comparing circadian times (CT) 0 and 8 we observed a similar fold change of U2af26 alternative splicing in the absence of external cues (DD) as in a standard LD setting. In addition, we have tested U2af26 splicing with the circadian clock in mouse liver as a second model for peripheral clocks (Figures S1J–S1L). We detected appearance of U2AF26DE67 in LD as well as DD conditions with the same acrophase and somewhat reduced amplitude compared to cerebellum. Interestingly, when we investigated U2af26 alternative splicing in the central clock (SCN), we observed only weak expression of U2AF26DE67, which was not subject to circadian regulation (Figure 1D). This suggests that U2af26 alternative splicing is not directly light sensitive in the SCN, but rather that signals from the SCN control this splicing switch in the periphery. These experiments together define exclusion of U2af26 exons 6 and 7 as a robust circadian splicing switch in mouse peripheral clocks. Skipping of U2af26 Exons 6 and 7 Generates a Domain with Homology to Drosophila TIM Circadian expression of U2AF26DE67 prompted us to characterize this isoform in more detail. Interestingly, exclusion of exons 6 and 7 induces a frameshift allowing translation past the canonical termination codon far into the 30 UTR. This circadian frameshift generates a C terminus of 143 new amino acids



Figure 1. Circadian Alternative Splicing of U2af26 Exons 6 and 7 in Peripheral Clocks

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representing more than 50% of the U2AF26DE67 protein (Figure 2A). Strikingly, BLAST searches revealed a substantial homology to the Drosophila protein TIM (Figure 2B; 28% identity, 48% positives, 67% tolerated), which, by controlling PER localization and stability, plays a central role in the fly circadian clock (Sehgal et al., 1994; Price et al., 1995; for comparison, homology TIM Drosophila/TIM mouse 17.5% identity; TIM Drosophila/ TIMEOUT mouse 34% identity). We note that especially amino acids 59–85 are almost completely conserved between ten Drosophila species (data not shown) and show particularly high sequence conservation with the U2AF26DE67 C terminus, suggesting an important role of this region. A mammalian Tim homolog has been described, but its function within the circadian clock is less well established (Barnes et al., 2003; Gotter, 2006). Circadian expression of the U2AF26DE67 C terminus, in the following referred to as TIM homology domain (THD), immediately opened the possibility of U2AF26 being a component of the mammalian clockwork. As a first step to investigate this hypothesis, we confirmed circadian U2AF26DE67 expression at the protein level. To this end, we raised an antibody against the THD and detected an immunoreactive protein species of the expected size whose expression profile resembles that of U2AF26DE67 mRNA (Figure 2C). This protein was absent in U2AF26-deficient mice (Figures S2A–S2C; see below for a

(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 U2AF26DE67 lacking exons 6 and 7. On the right, quantification of percent DE67 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 %DE67 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 %DE67 was performed as in (A) (n = 4; p < 0.0005). (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.

more detailed description of these animals) and absent in the presence of a blocking peptide, confirming specificity of the antibody (Figure S2D). Even though both U2AF26DE67 RNA and protein showed a peak at ZT8, the RNA stayed at a high level, whereas the protein already decreased at ZT12, suggesting the presence of further (post)translational regulation. Further characterization of U2AF26DE67 revealed almost exclusive cytoplasmic localization of the endogenous protein (Figure 2D) and a GFP-U2AF26DE67 fusion (Figure 2E, left). This is consistent with our earlier finding, that the U2AF26 nuclear localization signal (NLS) is encoded by exons 7 and 8 (Heyd et al., 2008). However, whereas GFP alone was distributed evenly between the nucleus and the cytoplasm (Figure 2E, right), GFPU2AF26DE67 completely lacks nuclear localization (Figure 2E), suggesting, in addition to the lacking NLS, active tethering to the cytoplasm. Accordingly, the region displaying homology to the THD falls within the cytoplasmic localization domain of TIM (Saez and Young, 1996), and the THD is sufficient for cytoplasmic localization (Figure S2E). As circadian expression requires fast protein turnover, we investigated the stability of U2AF26DE67 using the translation inhibitor Cycloheximide (CHX). To this end, we expressed GFP-tagged proteins in 293 cells, and their abundance was investigated at different time points after addition of CHX. U2AF26fl-GFP showed a half-life Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 3



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Figure 2. Skipping of U2af26 Exons 6 and 7 Generates a Domain with Homology to Drosophila TIM

(A) Scheme illustrating the exon-skipping (U2AF26DE67) induced frameshift resulting in translation far into the 30 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 U2AF26DE67. 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-U2AF26DE67 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-U2AF26DE67 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 U2AF26DE67. GFP-U2AF26DE67-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.

of over 24 hr; in contrast, U2AF26DE67 was dramatically destabilized with a half-life well below 3 hr regardless of N- or C-terminally attached GFP (Figures 2F, 2H, S2G, and S2H); this was not due to different localizations, as the cytoplasmic U2AF26DE7 (Heyd et al., 2008) was as stable as the nuclear U2AF26fl (Figure S2F). The loss of GFP-U2AF26DE67 was completely blocked by addition of the proteasome inhibitor MG132, which was not the case for inhibitors of the lysosome or the autophagosome. This strongly suggests a specific, proteasome-mediated degradation pathway (Figures 2G, 2H, and S2I). As for the cytoplasmic localization, the THD within U2AF26DE67 was sufficient for the destabilizing effect (Figure S2J). Endogenous U2AF26DE67 in WT liver cells also displayed fast turnover (Figure S2K), confirming our results from overexpressed GFP-U2AF26DE67. We therefore conclude that the circadian splicing switch leading to expression of the THD within U2AF26 enables cytoplasmic, circadian expression of this particular U2AF26 isoform. 4 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

U2AF26DE67 Interacts with and Specifically Destabilizes PER1 As TIM interacts with PER, we next investigated whether U2AF26DE67 has corresponding properties. Immunoprecipitations (IPs) of PER1-Flag showed an association with GFP-tagged U2AF26DE67 (Figure 3A, left) which was specific, as GFP alone did not precipitate (Figure 3A, right), and stable, as it was observed in the presence of up to 1.2M salt (Figure 3B). Conversely, we were able to coprecipitate PER1 with U2AF26DE67, confirming a complex formation (Figure 3C). Importantly, U2AF26DE67-PER1 complex formation was also observed in ZT8 liver cytoplasmic extracts from WT but not from U2AF26-deficient mice, confirming a specific complex with endogenous proteins (Figure 3D). This interaction was not mediated by the novel C terminus, but rather by the first zinc finger of U2AF26 (Figures 3E, 3F, and S3A). This region of U2AF26 shows perfect homology to U2AF35, and consistently,



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Figure 3. PER1 Interacts with U2AF26 via Its PasB Domain (A) Specific coprecipitation of U2AF26DE67 with PER1-Flag. Hek293 cells were cotransfected with GFP (right) or GFP-U2AF26DE67 (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 antiGFP 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-U2AF26DE67 is stable up to 1.2M NaCl and 2% TritonX. (C) PER1 coprecipitates with U2AF26DE67. Hek293 cells were cotransfected with MS2-Flag-U2AF26DE67 and PER1. Immunoprecipitations were performed as in (A). (D) Endogenous U2AF26DE67 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 (DCk1). See also Figure S3B. See also Figure S3.

we also observe an interaction between U2AF35 and PER1 (Figure 3G). Within PER1 the U2AF26-interacting region falls within the PasB domain (Figures 3H and S3B), which also mediates the interaction of TIM and PER in Drosophila (Hennig et al., 2009). Having confirmed a complex formation between U2AF26 and PER1, we asked whether this interaction would, as is known for the TIM-PER interaction, control turnover of PER1. When coexpressing PER1 and GFP-U2AF26DE67 in 293 cells we observed a strong decrease in PER1 half-life compared to GFP control (Figure 4A). Several lines of evidence demonstrate that this effect depends on the U2AF26/PER1 interaction and the presence of the novel C terminus: First, neither U2AF26fl, which interacts with PER1 but is stable itself, nor the THD alone, which does not interact with PER1 but is rapidly degraded, was sufficient

to alter PER1 stability (Figure 4A, left). Second, while the U2AF26-interacting PasB domain showed U2AF26DE67-dependent destabilization, this was not the case for the noninteracting C terminus of PER1 (DCK1 mutant; Figure 4B, right). Interestingly, this process was also proteasome dependent but was not blocked by MG132. Instead, Bortezomib, which preferentially inhibits a different activity of the proteasome (Kisselev et al., 2006), blocked U2AF26DE67-dependent PER1 degradation (Figure 4C). To confirm a U2AF26DE67-dependent control of PER1 stability at endogenous expression levels in a physiologically relevant setting, we compared PER1 stability in WT and U2AF26 KO liver. To this end, we treated liver cells isolated at ZT8 from both genotypes with CHX and investigated PER1 decay. In accordance with our overexpression studies, we observed significantly reduced PER1 stability in WT mice Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 5



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Figure 4. U2AF26DE67 Promotes Proteasomal Degradation of PER1 (A) PER1 half-life is specifically decreased by U2AF26DE67. 293 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) U2AF26DE67 selectively destabilizes the interacting PasB domain. PER1 deletion constructs (PasB or DCk1) were cotransfected either with GFP or with U2AF26DE67. 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-U2AF26DE67/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 U2AF26DE67-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.

(Figure 4D). The combined data suggest that U2AF26DE67 increases PER1 turnover, probably by facilitating its recruitment to the proteasome, but once there, both proteins are degraded independently of each other by different proteasomal activities. Consistent with conserved PasB domains in PER1 and PER2, U2AF26DE67 was also able to coprecipitate PER2 (Figure S4A). It is interesting to note that PER2 turnover, despite its interaction, was not increased by U2AF26DE67 (Figure S4B). Furthermore, we find that under our experimental conditions proteasome inhibitors MG132 or Bortezomib only weakly affect half-life of 6 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

PER2 (Figure S4B), suggesting that its turnover is at least partially proteasome independent. These data suggest a PER1-specific effect of U2AF26DE67, which could be explained by PER1 and PER2 stability being controlled by distinct mechanisms, with U2AF26DE67 only contributing to proteasome-dependent PER1 degradation. As PER1 and PER2 have overlapping yet distinct functions, e.g., in clock resetting (Albrecht et al., 1997), the specific effect of U2AF26DE67 on PER1 was suggestive of an involvement of U2AF26DE67 in PER1-controlled aspects of the circadian clock (see below).



U2AF26-Deficient Animals Show Defects in Circadian Gene Expression To analyze in vivo relevance of U2AF26DE67 controlled PER1 turnover, we investigated PER1 protein levels in WT and U2AF26-deficient mice during a circadian cycle in liver as a model for peripheral clocks. U2AF26-deficient mice (see Figure S2 for knockout strategy) are born at mendelian ratio, breed normally, and do not show any gross abnormalities. While we observed rhythmic PER1 expression with the expected phase and amplitude in WT mice (Yoo et al., 2013), PER1 expression became nearly arrhythmic in U2AF26deficient animals (Figures 5A and 5B). Consistent with U2AF26DE67-mediated destabilization of PER1, the drop of PER1 protein at ZT4 and ZT8 in WT mice coincides with the appearance of the U2AF26DE67 mRNA (Figure S1K) at these time points, which was confirmed at the protein level using liver cytoplasmic extracts (Figure 5C). In line with our finding that PER2 is not a direct target of U2AF26DE67-mediated degradation, PER2 protein expressions remained rhythmic in U2AF26-deficient animals (Figures S5A and S5B). In WT mice a drop in PER1 protein expression precedes an increase in mRNAs that are direct clock targets. Consistent with disturbed PER1 protein expression, we found the circadian expression of PER1 target genes such as Dbp, RevErba, or Per1 itself to be disturbed in liver and cerebellum of U2AF26-deficient animals (Figures 5D and 5E). The remaining rhythmicity of clock-controlled genes may indicate a partial redundancy between PER1 and PER2 such that circadian Per2 expression is sufficient to maintain an altered but rhythmic expression profile. To investigate other defects in U2AF26-deficient mice that could potentially contribute to a defect in the molecular clock and to address a potential function of the nuclear U2AF26fl isoform, we performed RNA-Seq of WT and KO brains. Despite using several analysis pipelines, we did not find obvious changes in the splicing profile between the two genotypes. For around 30 exons with a predicted moderate change in percent spliced in (PSI), we performed validations with our established radioactive RT-PCR protocol but failed to confirm significant differences. We furthermore used a targeted approach and compared splicing patterns of 25 exons known to be alternatively spliced in brain and/or with the circadian rhythm (Jensen et al., 2000; Gehman et al., 2011; McGlincy et al., 2012). As we did neither observe significant PSI differences in these experiments, our data so far suggest that loss of U2AF26 does not significantly alter splicing patterns in mouse brain. This somewhat surprising finding may indicate redundancy between U2AF26 and U2AF35 or compensatory effects—e.g., increased activity of U2AF35 upon deletion of U2AF26–with respect to splicing regulation; alternatively, we may have missed missplicing of mRNAs with low expression, or RNA-Seq of particular brain regions may be required to observe splicing alterations caused by U2AF26 deficiency. Altered Clock Resetting in U2AF26-Deficient Mice We then aimed to link defects in the molecular clock with altered behavior in U2AF26-deficient mice. Under 12 hr LD conditions U2AF26-deficient animals displayed wheel-running behavior

very similar to that of WT mice. Additionally we observe no strong alteration upon release into DD (Figures S6A and S6B). This result is consistent with the master clock being not strongly affected by loss of U2AF26, which is in line with weak and nonregulated U2AF26DE67 expression in the SCN. However, as PER1 is known to play a major role in clock resetting upon alteration of LD conditions in mice (Shigeyoshi et al., 1997; Albrecht et al., 1997; Akiyama et al., 1999; Sakamoto and Ishida, 2000; Jagannath et al., 2013), as is Drosophila TIM (HunterEnsor et al., 1996; Lee et al., 1996; Myers et al., 1996; Zeng et al., 1996; Li and Rosbash, 2013), we performed phaseadvance experiments. Animals were phase-advanced by 4 hr, and the time required to adapt to the new activity onset was measured. In accordance with earlier experiments, WT mice advanced their activity onset by 1 hr per day (Dudley et al., 2003). This adaptation was significantly faster in U2AF26deficient animals that changed their activity onset by over 3 hr on the first day already (Figures 6A and 6B). Interestingly, applying an 8 hr phase advance at CT16 in WT mice induced formation of U2AF26DE67 within 8 hr in particular in cerebellum, whereas this was not observed in liver (Figure 6C). This suggests that U2af26 alternative splicing is an early event during entrainment of specific peripheral clocks. Under these conditions Per1 mRNA expression was specifically deregulated 8 hr after applying the new LD rhythm in cerebellum but not in liver of U2AF26-deficient mice (Figure 6D). This provides a direct link between formation of the U2AF26DE67 isoform and regulation of the molecular clockwork, since we only observe differences between WT and KO animals in conditions, where the U2AF26DE67 isoform is present in WT. The time frame of U2AF26DE67 formation and the faster adaption to experimental jet lag of mice lacking U2AF26 is consistent with U2af26 alternative splicing being part of a feedback mechanism to limit an initial light-induced increase of Per1 in peripheral clocks (Figure S6C), thus providing a buffering system against sudden light changes. Finally, in order to provide evidence for crossspecies conservation of this regulatory mechanism, we investigated if a THD in an alternative reading frame of the U2af26 gene is present in other mammalian organisms. Interestingly, we found a highly conserved alternative reading frame in the rat U2AF26 30 UTR spanning amino acids 1–85 of the mouse U2AF26DE67 C terminus (Figures S6D–S6F), thus including the region that shows highest conservation in Drosophila Timeless (amino acids 59–85). As in mouse, this reading frame is used after exclusion of exons 6 and 7 (Figure S6D), and importantly, we observed regulation of U2af26 alternative splicing in rat cerebellum with a similar fold change as in mouse (Figure S6E), strongly pointing to a function that is conserved across species. We also noticed an alternative reading frame in human U2AF26 that shows reasonable homology to amino acids 61–143 of the mouse THD (Figures S6G– S6I). Usage of this frame depends on inclusion of exons 6 and 7 and the use of an alternative 30 splice site within exon 8 (Figure S6G). Sequencing PCR products of human U2af26 revealed the presence of a corresponding mRNA species (Figure S6H), further adding to the notion of crossspecies conservation. Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 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 U2AF26DE67-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.

DISCUSSION Our work provides the first evidence for a circadian splicing switch that controls rhythmic gene expression and behavior in 8 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

a mammalian system. In addition, we describe a molecular mechanism that reveals genomic information hidden in an alternative reading frame, in a supposedly untranslated part of the mRNA that is accessible by alternative splicing. This splicing switch



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 %DE67 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 U2AF26DE67 in light-induced control of PER1 protein and clock resetting. See also Figure S6.

within the U2af26 pre-mRNA generates a TIM homology domain and changes several key features of the protein. One consequence is a strongly reduced half-life of the U2AF26DE67 protein, enabling circadian expression. The fast, proteasomedependent degradation mediated by the THD is also conferred to interacting PER1, leading to a regulation of the molecular clockwork. Since different activities of the proteasome are required for U2AF26DE67 or PER1 degradation, U2AF26DE67 may serve to recruit PER1 to the proteasome, where both proteins are degraded by independent pathway. Another important difference between U2AF26 and U2AF26DE67 is the intracellular localization mediated by the THD. The exclusive cytoplasmic presence of U2AF26DE67 underlines the importance of protein localization for the molecular clockwork, as has been recently emphasized for CRY1 and CRY2 turnover (Yoo et al., 2013; Hirano et al., 2013). Although we have no evidence for the nuclear U2AF26fl in regulating PER1 stability or activity, the formation of an U2AF26-PER1 complex in the nucleus may have other,

as-of-yet-unidentified, roles in regulating PER1 or U2AF26 function. An additional layer of regulation could be added by the interaction of U2AF35 with PER1, which could compete with U2AF26DE67 for PER1 binding; however, as U2AF35 is predominantly nuclear, this would more likely affect the nuclear U2AF26-PER1 complex. An interesting question emanating from our work is the identification of the mechanism of splicing regulation. Expression of several splicing-regulatory proteins has been suggested to be regulated in a circadian manner (McGlincy et al., 2012), and proteins such as PSF and NONO that regulate alternative splicing in different contexts are known to be involved in clock regulation (Heyd and Lynch, 2010; Duong et al., 2011; Kowalska et al., 2013). However, splicing-regulatory proteins that regulate circadian splicing events in mammals and mechanisms that control their circadian activity have not been identified. As characterizing the precise mechanism of circadian U2af26 alternative splicing will enable the search for coregulated genes, it could Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc. 9



potentially yield a whole set of circadian exons controlled in a concerted manner. Such an approach complements the widely used RNA-Seq analyses and will substantially contribute to the understanding of circadian gene expression. Expression of the Timeless homology domain in the C terminus of U2AF26DE67 is regulated with the circadian rhythm but is also quickly entrainable by light. In fact, the kinetics of U2AF26DE67 formation begins to adapt fast after the first new light-on signal, suggesting a neuronal or humoral signal from the SCN to directly control U2af26 alternative splicing in cerebellum and potentially other brain regions. This fast adaptation and the direct influence of U2AF26DE67 on PER1 protein, which is known to be a key player in clock resetting, suggest a prominent role for U2af26 alternative splicing in connecting the circadian rhythm to changing external cues. While we observe this regulation in peripheral clocks, we cannot formally rule out that U2AF26 also plays a role in the SCN or the light input pathway that contribute to the jet lag phenotype of U2AF26-deficient mice. In contrast to TIM, which stabilizes PER in Drosophila (Price et al., 1995), U2AF26DE67 destabilizes PER1. However, as TIM is degraded and U2AF26DE67 is increased upon exposure to light, the result is in both cases a decreased stability/activity of PER protein in the hours following the light-on signal (Figure 6E). A notable difference is the kinetics of Timeless degradation within 1 hr (Hunter-Ensor et al., 1996; Lee et al., 1996; Myers et al., 1996; Zeng et al., 1996; Li and Rosbash, 2013) and U2AF26DE67 formation, which takes 4–8 hr. This is consistent with the much faster adaptation to altered LD cycles of flies versus mice (Dudley et al., 2003; Li and Rosbash, 2013); it may also reflect U2af26 alternative splicing not to be the resetting signal itself but rather being a negative feedback mechanism to an initial light-induced Per1 expression. Formation of U2AF26DE67 4–8 hr after light exposure and its negative effect on PER1 protein expression thus limit PER1 induction after phase shift in cerebellum and potentially other peripheral clocks, effectively slowing down the adaptation process, likely to allow a synchronous adaptation of the whole organism (model in Figure 6E). Intriguingly, a negative feedback mechanism involving Salt-inducible kinase-1 (Sik1) has just been proposed to limit Per1 mRNA induction upon phase shift in the SCN (Jagannath et al., 2013). Knocking down SIK1 in the SCN led to a much faster adaptation to experimental jet lag, whereas wheel-running behavior in constant darkness was only mildly affected, strongly resembling the phenotype of U2AF26-deficient mice. Both studies reveal mechanisms that in response to phase advance limit PER1 expression, either within 1–2 hr at the mRNA level in the SCN (Jagannath et al., 2013), or after 4–8 hr at the protein level in cerebellum (this study). These data provide molecular details and emphasize the importance of a buffering system in central and peripheral clocks that allows synchronized adaption to clock-resetting stimuli in order to prevent potentially pathogenic desynchronization. EXPERIMENTAL PROCEDURES Mouse Strains and Behavioral Assays U2AF26-deficient mice were generated by homologous recombination using standard techniques; a detailed cloning strategy is available upon request.

10 Molecular Cell 54, 1–12, May 22, 2014 ª2014 Elsevier Inc.

U2AF26-deficient animals were crossed into C57BL/6 background for more than 20 generations. All animal experiments were performed in accordance with institutional and governmental recommendations and laws. U2AF26 WT and deficient mice were housed under LD12:12 conditions unless otherwise mentioned. For preparation of RNA or protein, tissues were quickly removed and frozen in liquid nitrogen. SCN samples were prepared as described by Savelyev et al. (2011). For further analyses, tissues were homogenized either in PeqGold RNApure or in lysis buffer (Heyd and Lynch, 2010). Similarly, RNA from rat cerebellum was prepared from wild-type Wistar rats housed under LD12:12 conditions. For behavioral assays 8- to 14-week-old male mice were individually housed in cages equipped with running wheels and adapted for at least 10 days. Running wheels triggered reed switches that were connected to a Seeeduino Stalker, an Arduino compatible data-logging platform used to record activity. Free-running period measurements were based on at least 9-day activity recordings in constant darkness. For jet lag experiments, individually caged mice were entrained to a 12 hr LD cycle for at least 10 days before a 4 hr phase advance. The new activity onset was defined as six continuous 5 min bins with activity occurring after light-off. RNA Sequencing and Target Validation See Supplemental Experimental Procedures. Cell Culture, Transfections, and MEFs Hek293, HeLa, U2OS, and NIH 3T3 cells were maintained in DMEM medium containing 10%FBS and Pen/Strep (Invitrogen). Transfections of HeLa and Hek293 cells using Lipofectamine 2000 were done according to the manufacturer’s instructions. For the measurement of protein half-life, CHX (Sigma) was added 48 hr after transfection at a final concentration of 40 mg/ml. Inhibitors were used at the following concentrations: MG132 (10 mM, Biomol), Bortezomib (100 nM, Tinib-Tools), Bafilomycin A1 (1 mM, Sigma), and Leupeptine (10 mg/ml, Applichem). To determine the protein half-life in liver, we prepared liver single-cell suspensions from WT and U2AF26-deficient animals at ZT8. These were cultured in DMEM medium containing 20% FBS, Pen/Strep, and 40 mg/ml CHX. Primary mouse embryonic fibroblasts (MEFs) were prepared on day 13.5 and maintained in DMEM medium containing 20%FBS and Pen/Strep. For RNA isolation, 1 3 105 cells were seeded in 12-well plates in duplicate for each time point and grown until confluence. For Dexamethasone shock, cells were incubated in fresh medium containing 100 nM Dexamethasone for 1 hr. Afterward the medium was replaced by normal medium, and cells were harvested at the indicated times after shock. For Actinomycin D treatment, ActD (Sigma, 5 mg/ml) was added 12 and 20 hr postshock for another 4 hr. Constructs See Supplemental Experimental Procedures. RNA, RT-PCR, RT-qPCR RT-PCRs were done as previously described (Heyd and Lynch, 2010). RTqPCRs were performed using the ABsolute QPCR SYBR Green Mix (Thermo Fisher) on Stratagene Mx3000P instruments. See Supplemental Experimental Procedures for details. Western Blot, Immunoprecipitations, and Antibodies Lysates, nuclear/cytoplasmic fractionations (NX/CTX), western blots, and IPs were performed as previously described with minor adjustments (Heyd and Lynch, 2010). See Supplemental Experimental Procedures for details. For endogenous IPs, liver cytoplasmic extracts from single-cell suspensions were prepared as above. Extracts were precleaned for 1 hr using protein A/G Sepharose (Invitrogen). Afterward 100 mg of extract was incubated with either protein A/G Sepharose alone or protein A/G Sepharose coupled to an affinity-purified U2AF26DE67 antibody overnight in 500 ml Flag-lysis buffer (60 mM TrisHCl [pH 7.5], 30 mM NaCl, 1 mM EDTA, 1% Triton X-100, with proteinase inhibitors) containing 3% BSA. Beads were washed three times in Flag-lysis buffer, and IPs were analyzed by western blot. For details on antibodies used, see Supplemental Experimental Procedures.



SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and Supplemental Experimental Procedures and can be found with this article at http://dx.doi.org/10.1016/j. molcel.2014.04.015. AUTHOR CONTRIBUTIONS M.P. performed most experiments, with help from I.W., A.-S.S., M.M., and F.H. The wheel-running cages were assembled by F.F., who also collected activity data and performed bioinformatic analysis of RNA-Seq data. U2AF26 knockout design and generation of ESCs was done by F.H. in T.M.’s lab. Mice were generated and initially maintained in T.M.’s lab. F.H. designed and initiated the project; F.H. and M.P. planned experiments, analyzed data, and wrote the manuscript. ACKNOWLEDGMENTS

Dudley, C.A., Erbel-Sieler, C., Estill, S.J., Reick, M., Franken, P., Pitts, S., and McKnight, S.L. (2003). Altered patterns of sleep and behavioral adaptability in NPAS2-deficient mice. Science 301, 379–383. Duong, H.A., Robles, M.S., Knutti, D., and Weitz, C.J. (2011). A molecular mechanism for circadian clock negative feedback. Science 332, 1436–1439. Gehman, L.T., Stoilov, P., Maguire, J., Damianov, A., Lin, C.H., Shiue, L., Ares, M., Jr., Mody, I., and Black, D.L. (2011). The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain. Nat. Genet. 43, 706–711. Gekakis, N., Staknis, D., Nguyen, H.B., Davis, F.C., Wilsbacher, L.D., King, D.P., Takahashi, J.S., and Weitz, C.J. (1998). Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569. Gotter, A.L. (2006). A Timeless debate: resolving TIM’s noncircadian roles with possible clock function. Neuroreport 17, 1229–1233. Hennig, S., Strauss, H.M., Vanselow, K., Yildiz, O., Schulze, S., Arens, J., Kramer, A., and Wolf, E. (2009). Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2. PLoS Biol. 7, e94.

We thank Achim Kramer and Bert Maier (Humboldt University Berlin) for advice and helpful discussions, and Steve McKnight (UTSW, Dallas) for constructive comments on the manuscript. We also acknowledge Rolf Mu¨ller and Matthias Lauth (Philipps University Marburg) for sharing reagents; Ursula Koch and Elisabet Garcia-Pino (Free University Berlin) for help with SCN preparation; and Marie Hasselluhn for contributing as a rotation student. Work in F.H.’s lab is funded by an Emmy-Noether-Fellowship of the DFG (HE 5398/3-1); the present work was also supported by the University Hospital GiessenMarburg (UKGM).

Heyd, F., ten Dam, G., and Mo¨roÿ, T. (2006). Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing. Nat. Immunol. 7, 859–867.

Received: October 30, 2013 Revised: February 24, 2014 Accepted: April 9, 2014 Published: May 15, 2014

Hirano, A., Yumimoto, K., Tsunematsu, R., Matsumoto, M., Oyama, M., Kozuka-Hata, H., Nakagawa, T., Lanjakornsiripan, D., Nakayama, K.I., and Fukada, Y. (2013). FBXL21 regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes. Cell 152, 1106–1118.

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The Circadian Clock Gene Period1 Connects the Molecular Clock to Neural Activity in the Suprachiasmatic Nucleus.

The clock gene Period1 regulates innate routine behaviour in mice.

FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

A tunable artificial circadian clock in clock-defective mice.

The Arabidopsis sickle Mutant Exhibits Altered Circadian Clock Responses to Cool Temperatures and Temperature-Dependent Alternative Splicing.

An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action.

Rhythmic expression of circadian clock genes in the preovulatory ovarian follicles of the laying hen.

Neuronal circadian clock protein oscillations are similar in behaviourally rhythmic forager honeybees and in arrhythmic nurses.

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling.

Neurodegeneration and the Circadian Clock.

Regulation of the circadian clock through pre-mRNA splicing in Arabidopsis.

Natural selection against a circadian clock gene mutation in mice.

The circadian clock in immune cells controls the magnitude of Leishmania parasite infection.

Sec16 alternative splicing dynamically controls COPII transport efficiency.

Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

Physiology. Partitioning the circadian clock.

The spliceosome assembly factor GEMIN2 attenuates the effects of temperature on alternative splicing and circadian rhythms.

Circadian clock, cancer, and chemotherapy.

glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice.

Pharmacological control of circadian clock. Pharmacological manipulation of the circadian clock: from animal to human studies.

Alternative splicing generates novel Fads3 transcript in mice.