Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants

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Abstract
Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson–Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5′ splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence.