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

Vaughn JC, Ghosh S, Chen J - Open J Anim Sci (2013)

Bottom Line: We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.

ABSTRACT
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.

No MeSH data available.


Representative U4-/U6-snRNA secondary structures from phylogenetically diverse species, folded according to our general model. (a) H. sapiens; (b) S. cerevisiae; (c) T. thermophila; (d) P. falciparum; (e) D. discoideum; (f) A. thaliana; (g) C. reinhardtii; (h) T. brucei. Labeling is as in Figure 1.
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Figure 2: Representative U4-/U6-snRNA secondary structures from phylogenetically diverse species, folded according to our general model. (a) H. sapiens; (b) S. cerevisiae; (c) T. thermophila; (d) P. falciparum; (e) D. discoideum; (f) A. thaliana; (g) C. reinhardtii; (h) T. brucei. Labeling is as in Figure 1.

Mentions: The derived U4-/U6-snRNA duplex secondary structure model is shown in Figure 1, and structures from representative species at different taxonomic levels in Figures 2(a)–(h). A relatively large proportion of all nucleotides are base-paired in our U4-/U6-snRNA model. For example, in Drosophila 58% are base-paired in U4 and 63% in U6, whereas in the Y-shaped model the corresponding numbers are 58% and 33%. Four stem-loops (I-IV) are found to be present in the U4 structure for most species, so that our model both confirms and extends the secondary structure for free U4-snRNA previously proposed [32] using the phylogenetic approach with far fewer species. The existence of stem-loop IV in free U4-snRNA, proposed by the same authors, is also confirmed for all species studied by us. The overall conformation of the structure shown in our model is very similar in every species examined, with the exception of stem-loop III in U4-snRNA which is further discussed in Section 3.2. Each stem in our model has been proven by observation of numerous compensatory base mutations. Species within the flagellate group Euglenozoa were found to have the shortest overall U4- and U6-snRNA lengths (compare D. melanogaster in Figure 1 with T. brucei in Figure 2(h)). Despite the close similarity in conformation among species, nearly all stem lengths are however quite variable (Figure 1). The most consistent stem length is in U4 stem I, which ranges from 10 – 13 base pairs and is always interrupted by a structurally conserved bulge loop. A conspicuous highly conserved sequence tract in U4 is the putative SM-binding site, located near the 3’-end between stem-loops II and III, which matches the consensus sequence AU [4–6] G. Our study confirms the universality of the two major intermolecular base-paired zones of contact between the two RNA molecules (DS I and DS II) as originally proposed [12], with many examples of compensatory base mutations.


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

Vaughn JC, Ghosh S, Chen J - Open J Anim Sci (2013)

Representative U4-/U6-snRNA secondary structures from phylogenetically diverse species, folded according to our general model. (a) H. sapiens; (b) S. cerevisiae; (c) T. thermophila; (d) P. falciparum; (e) D. discoideum; (f) A. thaliana; (g) C. reinhardtii; (h) T. brucei. Labeling is as in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237228&req=5

Figure 2: Representative U4-/U6-snRNA secondary structures from phylogenetically diverse species, folded according to our general model. (a) H. sapiens; (b) S. cerevisiae; (c) T. thermophila; (d) P. falciparum; (e) D. discoideum; (f) A. thaliana; (g) C. reinhardtii; (h) T. brucei. Labeling is as in Figure 1.
Mentions: The derived U4-/U6-snRNA duplex secondary structure model is shown in Figure 1, and structures from representative species at different taxonomic levels in Figures 2(a)–(h). A relatively large proportion of all nucleotides are base-paired in our U4-/U6-snRNA model. For example, in Drosophila 58% are base-paired in U4 and 63% in U6, whereas in the Y-shaped model the corresponding numbers are 58% and 33%. Four stem-loops (I-IV) are found to be present in the U4 structure for most species, so that our model both confirms and extends the secondary structure for free U4-snRNA previously proposed [32] using the phylogenetic approach with far fewer species. The existence of stem-loop IV in free U4-snRNA, proposed by the same authors, is also confirmed for all species studied by us. The overall conformation of the structure shown in our model is very similar in every species examined, with the exception of stem-loop III in U4-snRNA which is further discussed in Section 3.2. Each stem in our model has been proven by observation of numerous compensatory base mutations. Species within the flagellate group Euglenozoa were found to have the shortest overall U4- and U6-snRNA lengths (compare D. melanogaster in Figure 1 with T. brucei in Figure 2(h)). Despite the close similarity in conformation among species, nearly all stem lengths are however quite variable (Figure 1). The most consistent stem length is in U4 stem I, which ranges from 10 – 13 base pairs and is always interrupted by a structurally conserved bulge loop. A conspicuous highly conserved sequence tract in U4 is the putative SM-binding site, located near the 3’-end between stem-loops II and III, which matches the consensus sequence AU [4–6] G. Our study confirms the universality of the two major intermolecular base-paired zones of contact between the two RNA molecules (DS I and DS II) as originally proposed [12], with many examples of compensatory base mutations.

Bottom Line: We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.

ABSTRACT
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.

No MeSH data available.