3962 Nucleic Acids Research, 2006, Vol. 34, No. 14 failed to identify proteins unique to the U12-type spliceosome in the C.elegans genome (1,58). Since other metazoans and plants have U12-type introns, it has been suggested that the C.elegans lineage lost the U12-type introns (8). Indeed, we found some vestigial U12-type C.elegans introns whose 50ss are strongly reminiscent of U12-type GT–AG introns in other species (Table 1).However, because the 30 end sequences of these introns have the signature features of C.elegans U2-type 30ss (see below), and a good candidate sequence for a U12-type BPS is missing, they are probably spliced by the U2-type machinery. Non-canonical U12-type introns. A signiﬁcant fraction of U12-type introns in our dataset ( 5%) lack the canonical pairs of dinucleotide (Table 2, see Materials and Methods). These transcripts are supported by RefSeq sequences. How- ever, a large fraction of these introns are incorrectly mapped in other genome browsers, probably due to automated map- ping based on canonical splice-site predictions. Only two of these introns were found in A.thaliana, includ- ing the previously described intron 7 of the AtG5 gene (59). Such non-canonical introns have been experimentally shown to be competent for splicing by the U12-type machinery (7). The most efﬁcient pairs were found to be GT–AG and AT– AC, but AT–AN (where N is any nucleotide) and GT–AT could also engage in productive splicing. Remarkably, the splicing efﬁciency of these splice-site combinations corre- lates with their frequencies in our database. Furthermore, evidence has been found for U12-type non-canonical splicing in samples from a Peutz–Jeghers syndrome patient that have a A to G mutation at the +1 position of the AT–AC U12-type 50 ss in the LKB1 gene (43). We have also identiﬁed another subtype of putative U12- type introns with GC–AG borders, which were predicted in an earlier survey (8). There are four candidates of these introns in the human and mouse, and one in the ﬂy. All of these candidates have a perfectly conserved GCATCCT 50 ss motif, and most have an optimal U12-type BPS at an appropriate location. Furthermore, the exonic nucleotide upstream of the 50ss of most of these candidates do not usu- ally conform to the U2-type consensus making them poor substrates for U2-type splicing. Biases in the distribution of U12-type and other rare introns. The persistence of U12-type introns over evolutionary time is a mystery, as they represent a very small fraction of all genes, and some lineages and organisms, such as C.elegans, appear Table 2. The distribution of non-canonical U12-type introns H.sapiens M.musculus D.melanogaster A.thaliana Total AT–AG AT–AA AT–AT AT–GT GT–AT GT–GG GT–TG GC–AG Total % of all U12-type 11 3 6 0 4 5 0 4 33 4.92 8 4 6 1 3 3 1 4 30 4.80 0 0 0 0 0 0 0 1 1 5.56 1 1 0 0 0 0 0 0 2 1.05 20 8 12 1 7 8 1 9 66 to have completely lost them. We used GObar (48) (see http://katahdin.cshl.org:9331/GO/GO.cgi), a tool that exploits the information in Gene Ontology to unveil pathways or functions that are enriched in a particular sample gene set, to study the functional distribution of genes containing U12-type introns. We ﬁnd that U12-type introns are usually found in genes involved in ion transport (25,60), protein trafﬁcking and cell-cycle control, among other processes. Genes with more than one U12-type intron are found more often than expected from a random model for the disribution of U12-type introns in genes, consistent with previous reports (6,8). H.sapiens and M.musculus have 45 and 46 genes that contain two U12-type introns, respectively, A.thaliana has only six, and D.melanogaster has none. Furthermore, human and mouse have two and three genes, respectively, with three U12-type introns, all of them members of the solute carrier family (SLC) gene family, that encode sodium and hydrogen exchanger proteins. In mammals, considering an average number of eight introns per gene (61) and assum- ing an independent U12-intron distribution model, the expected number of genes with two U12-type introns is only six. If we include more realistic distributions of introns amongst genes, the total expected goes up to only 16. This physical clustering of U12-type introns could be related to function or to the origin of U12-type introns. The U12-type machinery is 100 times less abundant in the nucleus than the U2-type machinery (62,63) and a possible consequence of this is slower splicing kinetics of U12-type introns. Indeed, there is some experimental corroboration for this idea (64). These observations suggest that U12-type introns persist because of their slower rates of splicing reac- tion, which might be important for the expression of the gene that contains them. If the U12-type intron is the slowest step in the pre-mRNA processing, having more than one U12-type intron in a gene might have no additional impact on splicing dynamics. The possibility of intron conversion from U12- to U2-type was previously demonstrated by looking at orthologous U12-type introns from different species (8,60), but the rate of conversion is unknown. As pointed out in these studies, the high conservation of U12-type splice sites and the high degeneracy of U2-type splice sites suggests that this con- version could be achieved by a few mutations, and that the reciprocal conversion would be very difﬁcult. Interestingly, we noticed frequent subtype switching of non-canonical U12-type introns between human and mouse. Out of 24 con- served U12-type introns between human and mouse of which at least one of the two is non-canonical, only 10 showed the same intron borders (Table 3) The remaining 14 introns underwent subtype switching. Amongst these, we found two cases of subtype switching from a non-canonical intron pair to another one, and ten introns that were switched from a non-canonical intron pair to a canonical GT–AG or AT–AC intron or vice versa. Remarkably, all these non-conserved intron borders differ at only one position. The AT–AG U12-type introns might represent viable inter- mediates of subtype switching events between U12-type AT–AC and GT–AG introns. In light of this, the >50% sub- type switching for the non-canonical introns seem to be at odds with the observation that no subtype switching between