Fbxl21 in the Circadian System (nt), giving a protein of 429 amino acids (Fig 1A/B). Overall sequence homologies to bovine, murine and human Fbxl3 were all .95%, with 100% amino acid conservation in the region 332– 385, which encodes a CRY binding domain (CBD, Fig 1G). Additionally, we isolated a truncated in-frame splice-variant (oFbxl3sv, GenBank EF643524), in which nt 209 to 354 are deleted (Fig 1A). Alternative splicing (AS) describes the process of splicing exons of pre-mRNAs in different arrangements and drastically expands the potential repertoire of protein variants [27– 30]. Since there is an evolutionary trend towards increased AS in complex organisms, this mechanism may resolve the paradoxical lack of increasing number of expressed genes in increasingly complex eukaryotic organisms [27–28]. It appears that AS is widespread since recent large-scale analyses indicate that as much as 70–80% of genes in mammals are subject to AS [29,31]. In the case of oFbxl3sv, AS spares the open reading frame but leads to a truncation of the F-box motif in the protein (Fig 1B, Pfam score diminished by 2.104-fold compared to oFbxl3fl), while the CBD is left intact. Such a protein is therefore predicted to have intact CRY binding while coupling with the proteasome degradation pathway may be impaired. We designed primers to encompass the predicted AS region (Fig 1A, O47C/O48C), or to select for the splice-variant (oFbxl3sv) (Fig 1A, O51C/O52C, see M&M for oligos sequences and details), and screened for Fbxl3 expression in ovine tissues (Fig 1C). This clearly demonstrates that both oFbxl3fl and oFbxl3sv are ubiquitously expressed. Sequence phylogeny analysis shows that Fbxl21 is the closest relative of Fbxl3 [26]. We therefore cloned a full-length cds for ovine Fbxl21 (oFbxl21fl, GenBank EU239380). This yielded a 1305 nt sequence giving a protein of 434 amino acids with a greater degree of inter-species divergence than was the case for Fbxl3 (94, 84 and 82% homology with bovine, mouse and human Fbxl21 respectively, Fig 1D). Fbxl21 appears to be a paralogue of Fbxl3 that arose through gene duplication relatively recently; indeed, unlike Fbxl3, no clear orthologue of Fbxl21 could be identified in fish genomes (Ensembl database). Within the region corresponding to the CBD of Fbxl3, amino acid identity to mFbxl21 was only 79% although most substitutions were either conservative or semi-consevative (Fig 1D/E and G). Here too, a splice-variant (oFbxl21sv, GenBank EU239381) was isolated. This splice-variant lacks 70 nt, is therefore out of frame (Fig 1D/E; fs: frame-shift) and presents an early stop-codon (TGA). This Figure 1. Cloning and mRNA distribution of ovine Fbxl3 and Fbxl21. A/D. Schematics depicting the cds of mRNA of Fbxl3 and Fbxl21, respectively. Primers used for RT-PCR are indicated. Note that Fbxl3sv is an in-frame splice-variant while Fbxl21sv is an out-of-frame splice-variant (fs for frame-shift) with an early stop-codon (TGA, nt 489). B/E. Schematics depicting the proteins encoded by the Fbxl3 and Fbxl21 transcripts, respectively. Note that part of F-Box motif of Fbxl21sv is disrupted due to the splicing event while the CBD is spared. Note also that Fbxl21sv is a short protein product of 171 amino acids with the last 8 amino acids (indicated in green) differing from Fbxl21fl due to the AS event. The F-Box motif is left intact but Fbxl21sv does not have a CBD. C. RT-PCR for Fbxl3 on central and peripheral tissues sampled at ZT4. Co-amplification of a CKId fragment was used as an internal positive control (oCKId, GenBank EF643522), absence of cDNA in the PCR mix as a negative control. Upper panel: screening with O47C/O48C primers. Lower panel: screening with O51C/O52C primers clarifies the tissue distribution of Fbxl3sv. F. RT-PCR for Fbxl21 on central and peripheral tissues sampled at ZT4. G. Sheep - mouse amino acid conservation within the CBD of Fbxl3 and Fbxl21. Residues associated with the Afterhours (Afh) and Overtime (Ovtm) Fbxl3 mutants are highlighted in red. Blue shading indicates residues that differ in at least one of the four sequences. Star, two dots and one dot indicate identical, conservative and semi-conservative substitutions, respectively. Pfam scores for F-box motifs: Fbxl3fl 3.9e209, Fbxl3sv 2.1e205, Fbxl21fl and Fbxl2sv 6.5e211. doi:10.1371/journal.pone.0003530.g001 PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3530