Micro-exons are exons of very small size (usually 3-30 nts). Some micro-exons are alternatively spliced. Their functions, regulation and evolution are largely unknown. Here, we present an example of an alternatively spliced 3 bp micro-exon (micro-Ex8) in the homothorax (hth) gene in Drosophila. Hth is involved in many developmental processes. It contains a MH domain and a TALE-class homeodomain (HD). It binds to another homeodomain Exd via its MH domain to promote the nuclear import of the Hth-Exd complex and serve as a cofactor for Hox proteins. The MH and HD domains in Hth as well as the HTh-Exd interaction are highly conserved in evolution. The alternatively spliced micro-exon lies between the exons encoding the MH and HD domains. We provide clear proof that the micro-Ex8 is produced by alternative splicing from a 48 bp full-length exon 8 (FL-Ex8) and the micro-Ex8 is the first three nt is FL-Ex8. We found that the micro-Ex8 is the ancient form and the 3 + 48 organization of alternatively spliced overlapping exons only emerged in the Schizophora group of Diptera and is absolutely conserved in this group. We then used several strategies to test the in vivo function of the two types of isoforms and found that the micro-Ex8 and FL-Ex8 isoforms have largely overlapping functions but also have non-redundant functions that are tissue-specific, which supports their strong evolutionary conservation. Since the different combinations of protein interaction of Hth with Exd and/or Hox can have different DNA target specificity, our finding of alternatively spliced isoforms adds to the spectrum of structural and functional diversity under developmental regulation. In eukaryotes, most genes contain exons that are separated by non-coding introns. Mature mRNAs are formed from primary transcripts by excising introns and splicing exons together. Pre-mRNA splicing is conducted by the spliceosome that recognizes the consensus motifs including the 5′ splice site GU, the branch point followed by a short polypyrimidine tract and the 3′ splice site AG 1,2. The spliceosome is a large ribonucleoprotein complex comprised of five small nuclear ribonucleoproteins (snRNPs, U1, U2, U4/U6, and U5) with small nuclear RNAs (snRNAs) and a number of associated proteins 3. Alternative splicing confers the flexibility of the pre-mRNA splicing process, and is considered an important trigger for proteome expansion and gene diversity during evolution 4,5. Exons of very small size (usually 3-30 nts) are called micro-exons 6,7. Examples include a 6 bp exon in the invected gene in Drosophila 8 ,a 9 bp exon in wheat 1-SST gene 9, a 9 bp exon in potato invertase genes 10 ,a 1 bp exon in Arabidopsis APC-11 gene 11 ,a 3 bp exon in Drosophila troponin T gene 12 ,two 5 bp exons in Drosophila Ubx gene 6 ,two 9 bp exons in Drosophila Fasciclin 1 gene 13 ,an alternatively spliced 3 bp exon in mouse and rat NCAM gene 14,15 ,a 6 bp exon in chicken cardiac troponin T gene 16 16. Special algorithms were developed to systematically identify the micro-exons, especially...
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