transcript yields a predicted protein of only 171 amino acids, leaving the F-box motif intact but lacking the CBD (Fig 1D/E). Such a protein is therefore unlikely to bind CRY proteins while coupling with the proteasome degradation pathway would be retained. As for oFbxl3, we designed primers (O49C/O50C) to determine the tissue distribution of oFbxl21fl and oFbxl21sv. This revealed that Fbxl21 exhibited much more varied levels of expression between tissues than Fbxl3, with maximal expression in the adenohypophysis, hypothalamus and pineal (all neuroen- docrine structures associated with timing and homeostasis) and no expression evident in liver, adrenal or white adipose tissue (WAT) (Fig 1F). The truncated transcript, oFbxl21sv, was detected in all tissues in which oFbxl21fl was also expressed. A similar distribution pattern was observed when RNA were extracted from animals sampled at either ZT4 (present data) or ZT15 (data not shown) indicating that absence of detection of Fbxl21 in liver, adrenal and WAT is unlikely due to a time-of-day effect on sampling (see below). Transcripts encoding truncated protein products such as oFbxl21sv have long been considered as erroneous splicing events but recent evidence indicates they may have specific functions [27,30,32]. Strikingly, conservation in splice-variant isoforms between mouse and human is very low, with estimates around 10–20% [28–29]. This might indicate meaningful evolutionary differences or, alternatively, reveal that splice-variants are merely a reflection of molecular noise generated by the splicing machinery [28–30]. In this context it is noteworthy that the same in-frame Fbxl3sv transcript characterized in the sheep is also present in the mouse (data not shown). Furthermore, we cloned a novel splice- variant for Fbxl21 in the mouse that encodes for a truncated protein product of a sequence reminiscent to that of oFbxl21sv (GenBank EU770600). These data demonstrate that AS is commonplace in the F-box protein family as emphasized elsewhere [26], which offers another putative layer of fine-tuning in the circadian clock. Furthermore, the fact that oFbxl3fl and oFbxl3sv are ubiquitously expressed while oFbxl21fl and oFbxl21sv display a clear-cut tissue-specific distribution strength- ens the view that circadian clocks among tissues might have different characteristics, as previously suggested [7,33]. Fbxl3 and Fbxl21 physically associate with Cry1 We used co-immunoprecipitation assays to determine whether sheep Fbxl3/21 orthologues bind to CRY1 in-vitro. We therefore cloned a full-length cds for ovine Cry1 (oCry1, GenBank EF494243) in a Flag-tagged expression vector while Fbxl3fl, Fbxl3sv, Fbxl21fl and Fbxl21sv were cloned in Myc-tagged Fbxl21 in the Circadian System expression constructs. These were co-transfected into COS7 cells in different combinations, prior to co-immunoprecipitation with beads coated with a-Myc antibodies followed by Western blotting (WB) with a-Myc or a-Flag antibodies (WB, Fig 2A). Consistent with data in the mouse [23,25], we observed pull-down of oCRY1 by oFbxl3fl. This was also observed for oFbxl3sv, as predicted by the preservation of the CBD in this transcript. A similar pull-down was observed for oFbxl21fl, but not for oFbxl21sv that lacks the CBD. These findings confirm predictions and indicate that interaction with CRY1 requires the CBD. In spite of the sequence divergence between the Fbxl21 CBD of sheep and mouse (Fig 1G), pull-down experiments using mFbxl21 instead of oFbxl21 demonstrate that the CRY1 binding ability is intact (Fig 2B). Differential effects of ovine Fbxl variants on CRY1 protein degradation We then assessed whether oFbxl proteins affect the degradation kinetics of molecular clock components. For this purpose full- length cds for orthologues of Clk (oClk, GenBank EU016223) and Bm1 (oBm1, GenBank EF494245) were cloned in the sheep. COS7 cells were co-transfected with oCry1, oBm1 and oClk in combination with the three Fbxl constructs which co-immuno- precipitated with oCRY1, and then treated with the protein synthesis inhibitor cycloheximide (CHX, 25 mg/ml) for 0–8 h. Proteins were extracted and relative levels were assessed by WB (Fig 3A). The Fbxl proteins had different effects on baseline oCRY1 and oBM1 levels immediately prior to CHX treatment (t = 0), but no impact on those for oCLK (Fig 3B). Full-length oFbxl3 strongly reduced baseline of oCRY1 levels while oFbxl21fl did not have any clear effect on oCRY1 levels, in agreement with previous results (see Fig 2A, input). Contrastingly, baseline levels of oCRY1 showed a trend towards higher levels by co-transfection of oFbxl3sv, in-line with the predicted impaired coupling to the proteasome degradation pathway and suggestive of a dominant-negative effect. To normalise for these differing baseline levels, data were replotted as a fraction of t = 0 values for each expression vector combination (Fig 3C). Degradation rates in cells co-transfected with empty vector (ev) served as controls. These data demonstrate that only the rate of oCRY1 degradation is highly dependent on co-transfection of oFbxl expression vectors (two-way ANOVA, the value for the interaction treatment6time is indicated on the right of Fig 3C). When compared to the ev control condition it was clear that oFbxl3fl exerts the strongest degradation promoting effect on CRY1 proteins (post-hoc test, p,0.01), reducing their Figure 2. Ovine and murine Fbxl3 and Fbxl21 physically associate with oCry1. A. Extracts from COS7 cells co-transfected with Myc-Fbxl and Flag-Cry1 expression vectors were submitted to immunoprecipitation (IP) with a-Myc beads followed by western-blotting (WB). The oCRY1 protein was readily co-purified with oFbxl3fl, oFbxl3sv and oFbxl21fl but not with oFbxl21sv. Note the low CRY1 levels in the input when Fbxl3fl was present. Similar detection levels of IgG (from Myc antibodies) across all conditions demonstrate loading of comparable amounts of supernatant. ev = empty expression vector. B. The use of similar methods demonstrates that the mouse Fbxl21 also interacts with oCRY1. doi:10.1371/journal.pone.0003530.g002 PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3530