Most of the eukaryotic protein-coding sequences are interrupted by multiple non-coding sequences, introns, which are excised from the transcript through the action of a complex molecular machine, the spliceosome, which consists of snRNP (small nuclear ribonucleoprotein particles) and hnRNPs (heterogeneous nuclear ribonucleoprotein particles), and is conserved throughout the eukaryotic world 123. There are two types of introns, U2 and U12, which are excised by distinct spliceosomes, the major and the minor one, respectively; over 99% of the eukaryotic introns belong to the U2 class, and the remaining ones comprise the U12 class 456.

The atypical U12 introns have been recognized through their unusual splice junction structure, namely, |AT at the donor splice site and AC| at the acceptor splice site 78. A closer examination of the sequences of these introns revealed additional features that distinguish them from the major U2 introns, including conservation of unusual signals at the donor splice site (|ATATCCTT) and in the vicinity of the acceptor splice site (TCCTTAAC 10–15 bases upstream of the splice junction). Subsequently, it has been shown that some |GT-AG| introns are also spliced out by the U12 spliceosome; as it turns out, actually, the majority of U12 introns are of the |GT-AG| type 9. The U12 spliceosome was first identified and characterized in animals, where it has been shown to contain several unique small nuclear RNAs (snRNAs), including the eponymous U12, U11, U4atac, and U6atac, that are structurally similar and, apparently, functionally analogous to the snRNAs of the major spliceosome 101112. Recently, RNA and protein components of the minor spliceosome along with U12 introns have been also identified in plants, fungi, and unicellular eukaryotes 1314. Thus, the minor spliceosome and U12 introns that it removes have been detected in representatives of all eukaryotic supergroups for which substantial amounts of genome sequences are available; so it appears that the minor splicing system is as ancient as the major one 414.

Comparative analyses of the gene structures in orthologous genes from diverse eukaryotes have shown that up to 30% of U2 intron positions are conserved between animals and plants 1516. Combined with the demonstration that parallel gains of introns in the same position could account only for a relatively small fraction (~10%) of shared plant and animal intron positions 1718, these findings indicate that a substantial fraction of introns in intron-rich extant genomes descends from the earliest stages of eukaryotic evolution.

The positions of U12 introns tend to be conserved among vertebrates 419, and two shared U12 intron positions have been detected in animal and plant genes for Na⁺/H⁺ antiporters 20. However, the overall level of conservation of U12 introns between plants and animals, and hence the depth of the evolutionary conservation of U12 introns is not known. We analyzed the available data on U12 introns in human and Arabidopsis thaliana genomes in order to systematically compare their conservation with that of U2 introns.

The U12 intron sequences were extracted from the SpliceRack database 21, and Arabidopsis and human genomic sequences were collected from NCBI. The U12 introns were mapped onto the genomic sequences yielding 570 human and 182 Arabidopsis U12 validated intron positions available for comparative analysis (Additional file 1). Probable orthologs were identified among human and Arabidopsis genes containing U12 introns by BLAST comparison, and the intron positions were mapped onto aligned protein sequences as previously described (16 and Additional file 1). This procedure yielded 133 pairwise alignments of human-Arabidopsis orthologs with a total of 1796 intron positions (935 human and 861 Arabidopsis). Of these intron positions, 155 were conserved including 20 U12, 115 U2, and 20 "mixed" positions, with a U12 intron in one species and a U2 intron in the other. The fraction of shared intron positions was close to the previous estimates 1618. In agreement with the results previously reported for U2 introns 17, simulation of the intron distribution in the analyzed set of orthologous genes by random intron shuffling (10,000 simulations) among the identified intron positions showed that the probability to observe 20 U12 intron positions shared by human and Arabidopsis genes as a result of independent inrons gains is < 0.0001. Thus, the shared U12 introns, primarily, reflect bona fide evolutionary conservation. Moreover, and unexpectedly, the fraction of conserved U12 intron positions in the analyzed set of human/Arabidopsis orthologs was significantly greater than the fraction of conserved U2 introns (Table 1).

Among the "mixed" positions, 15 contain U12 introns in the human genes opposite a U2 intron in Arabidopsis, and only 5 contains a U2 intron in the human gene opposite U12 in Arabidopsis. This significantly asymmetric distribution of the U12-U2 mixed sites (P = 0.02 according to the binomial test) is likely to reflect intensive U12 to U2 conversion in plant evolution which might be the reason behind the small number of U12 introns in Arabidopsis compared to humans.

It has been noticed previously that genes of intron-poor organisms display a substantial bias of intron distribution over the coding sequence length, with introns strongly over-represented in the 5'-portion of the genes, an observation that suggests a strong preference for intron loss in the 3'-portions of genes 2223. Moreover, even in intron-rich genomes, highly conserved, ancient introns concentrate in the 5'-portions of genes, suggesting the possibility of their preferential involvement in expression regulation and, possibly, other functional roles 24. The distribution of U12 introns, in a sense, emulates the overall distribution of introns in intron-poor genome because there are so few representatives of this class of introns in any of the sequenced genomes. We, therefore, compared the distributions of U12 and U2 introns across the lengths of the coding sequences of human and Arabidopsis genes. As shown in Figure 1, in both organisms, the U12 introns show substantially greater enrichment in the 5'-portions of genes than the U2 introns. An even more notable observation was made when we compared the partitioning of conserved U12 intron positions and the mixed positions. There was a dramatic excess of conserved U12 intron positions in the 5'-portions of the analyzed genes and a reciprocal excess of apparent U12 to U2 conversions in the 3'-portions of Arabidopsis genes (Table 2).

Although the number of U12 introns is small, calling for some caution in the interpretation of the results, taken together, these findings are compatible with the notion that U12 introns in 5'-portions of animal and plant genes tend to be conserved owing to their functional importance. It has been shown that the rate of removal of U12 introns from the respective transcripts by the minor spliceosome is several-fold slower than the rate of removal of U2 introns by the major spliceosome, leading to the hypothesis that U12 introns down-regulate the expression of their host genes 525. The findings described here add credence to this hypothesis and suggest that recruitment of U12 introns for this regulatory role might account for their notable evolutionary conservation, and for the fact that U12 introns linger in numerous eukaryotic genome despite the ongoing, apparently, unidirectional conversion into U2.

The authors declare that they have no competing interests.

MKB performed sequence comparisons and contributed to the analysis of the results; WM contributed to the interpretation of the results; IBR contributed to the analysis of the results and wrote the initial draft of the manuscript; EVK incepted the study, contributed to the analysis of the results and wrote the final manuscript; all authors edited and approved the final version.