Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively l...Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5’-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5’-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.展开更多
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 assembl...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-4Fto a5’-UTR regulatory region within its own pre-mRNA, which may play a role in negative feedback control.展开更多
Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes a...Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.展开更多
文摘Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5’-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5’-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.
文摘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-4Fto a5’-UTR regulatory region within its own pre-mRNA, which may play a role in negative feedback control.
文摘Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.
