A phylogenetic study of Drosophila splicing assembly chaperone RNP-4F associated U4-/U6-snRNA secondary structure

The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4- and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4- or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding site for Drosophila splicing assembly factor RNP-4F to a 5'-UTR regulatory region within its own premRNA, which may play a role in negative feedback control.