RESUMO
Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11 phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation) and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.
RESUMO
Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteômica , Humanos , Códon/genética , Códon/metabolismo , Ribossomos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biossíntese de ProteínasRESUMO
Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Replicação do DNA , DNA Fúngico/metabolismo , Neurospora/metabolismo , Nucleossomos/metabolismo , Animais , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/genética , Histonas/metabolismo , Neurospora/genética , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Relação Estrutura-Atividade , Fatores de Tempo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain upstream of the cat-3 gene, hereby called 5H-cat-3 domain, to constrain aberrant heterochromatin spreading. Interestingly, aberrant spreading of the 5H-cat-3 domain observed in the dmm-1KO strain is accompanied by robust deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5H-cat-3 domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5H-cat-3 domain. Importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin. Together, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.
Assuntos
Heterocromatina , Histonas , Neurospora crassa/metabolismo , Metilação de DNA , Eucromatina/genética , Estudo de Associação Genômica Ampla , Heterocromatina/genética , Histonas/genética , Histonas/metabolismoRESUMO
Codon usage bias is a fundamental feature of all genomes and plays an important role in determining gene expression levels. The codon usage was thought to influence gene expression mainly due to its impact on translation. Recently, however, codon usage was shown to affect transcription of fungal and mammalian genes, indicating the existence of a gene regulatory phenomenon with unknown mechanism. In Neurospora, codon usage biases strongly correlate with mRNA levels genome-wide, and here we show that the correlation between codon usage and RNA levels is maintained in the nucleus. In addition, codon optimality is tightly correlated with both total and nuclear RNA levels, suggesting that codon usage broadly influences mRNA levels through transcription in a translation-independent manner. A large-scale RNA sequencing-based genetic screen in Neurospora identified 18 candidate factors that when deleted decreased the genome-wide correlation between codon usage and RNA levels and reduced the codon usage effect on gene expression. Most of these factors, such as the H3K36 methyltransferase, are chromatin regulators or transcription factors. Together, our results suggest that the transcriptional effect of codon usage is mediated by multiple transcriptional regulatory mechanisms.
Assuntos
Uso do Códon/genética , Neurospora crassa/genética , RNA Mensageiro/biossíntese , Transcrição Gênica , Cromatina/genética , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico/genética , RNA Mensageiro/genéticaRESUMO
In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism.
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Genes Fúngicos/genética , Neurospora crassa/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exonucleases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Mutação , Ligação Proteica , Estabilidade de RNA/genéticaRESUMO
Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy, and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance the rate of translation elongation, whereas non-optimal codons slow elongation. Codon usage also controls ribosome traffic on mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with template mRNAs designed to increase the signal-to-noise ratio. Finally, we demonstrate that codon usage regulates protein function by affecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding.
Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica , Dobramento de Proteína , Animais , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Modelos Moleculares , Neurospora crassa/genética , Neurospora crassa/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.
Assuntos
Anticódon/genética , Uso do Códon , Elongação Traducional da Cadeia Peptídica/genética , Proteoma/genética , RNA de Transferência de Arginina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Anticódon/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inosina/metabolismo , Neurospora crassa/genética , RNA de Transferência de Arginina/metabolismo , Ribossomos/metabolismoRESUMO
Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.
Assuntos
Proteínas de Drosophila/genética , Fatores de Terminação de Peptídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Sequência de Aminoácidos/genética , Animais , Códon sem Sentido/genética , Códon de Terminação/genética , Drosophila/genética , Neurospora/genéticaRESUMO
KRAS and HRAS are highly homologous oncogenic Ras GTPase family members that are mutated in a wide spectrum of human cancers. Despite having high amino acid identity, KRAS and HRAS have very different codon usage biases: the HRAS gene contains many common codons, and KRAS is enriched for rare codons. Rare codons in KRAS suppress its protein expression, which has been shown to affect both normal and cancer biology in mammals. Here, using HRAS or KRAS expression in different human cell lines and in vitro transcription and translation assays, we show that KRAS rare codons inhibit both translation efficiency and transcription and that the contribution of these two processes varies among different cell lines. We observed that codon usage regulates mRNA translation efficiency such that WT KRAS mRNA is poorly translated. On the other hand, common codons increased transcriptional rates by promoting activating histone modifications and recruitment of transcriptional coactivators. Finally, we found that codon usage also influences KRAS protein conformation, likely because of its effect on co-translational protein folding. Together, our results reveal that codon usage has multidimensional effects on protein expression, ranging from effects on transcription to protein folding in human cells.
Assuntos
Códon/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Acetilação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cofilina 2/genética , Regulação da Expressão Gênica , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Metilação , Conformação Proteica , Dobramento de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , RNA Mensageiro/metabolismo , Temperatura , Iniciação da Transcrição GenéticaRESUMO
Codon usage biases are found in all eukaryotic and prokaryotic genomes, and preferred codons are more frequently used in highly expressed genes. The effects of codon usage on gene expression were previously thought to be mainly mediated by its impacts on translation. Here, we show that codon usage strongly correlates with both protein and mRNA levels genome-wide in the filamentous fungus Neurospora Gene codon optimization also results in strong up-regulation of protein and RNA levels, suggesting that codon usage is an important determinant of gene expression. Surprisingly, we found that the impact of codon usage on gene expression results mainly from effects on transcription and is largely independent of mRNA translation and mRNA stability. Furthermore, we show that histone H3 lysine 9 trimethylation is one of the mechanisms responsible for the codon usage-mediated transcriptional silencing of some genes with nonoptimal codons. Together, these results uncovered an unexpected important role of codon usage in ORF sequences in determining transcription levels and suggest that codon biases are an adaptation of protein coding sequences to both transcription and translation machineries. Therefore, synonymous codons not only specify protein sequences and translation dynamics, but also help determine gene expression levels.
Assuntos
Códon , Regulação da Expressão Gênica , Transcrição Gênica , Composição de Bases , Genoma Fúngico , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Neurospora/genética , Neurospora/metabolismo , Biossíntese de Proteínas/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Cytosine methylation of DNA is an important epigenetic gene silencing mechanism in plants, fungi, and animals. In the filamentous fungus Neurospora crassa, nearly all known DNA methylations occur in transposon relics and repetitive sequences, and DNA methylation does not depend on the canonical RNAi pathway. disiRNAs are Dicer-independent small non-coding RNAs that arise from gene-rich part of the Neurospora genome. Here we describe a new type of DNA methylation that is associated with the disiRNA loci. Unlike the known DNA methylation in Neurospora, disiRNA loci DNA methylation (DLDM) is highly dynamic and is regulated by an on/off mechanism. Some disiRNA production appears to rely on pol II directed transcription. Importantly, DLDM is triggered by convergent transcription and enriched in promoter regions. Together, our results establish a new mechanism that triggers DNA methylation.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Interferência de RNA , Pequeno RNA não Traduzido/genética , DNA Fúngico/genética , Inativação Gênica , Mutação , Neurospora crassa/genética , Sequências Repetitivas de Ácido Nucleico/genética , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction.
Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Fosforilação , Precursores de RNA/metabolismo , Precursores de RNA/genética , Células HeLa , Sítio de Iniciação de TranscriçãoRESUMO
Cells utilize different metabolic processes to maintain their growth and differentiation. Tumor cells have made some metabolic changes to protect themselves from malnutrition. These metabolic alterations affect the tumor microenvironment and macroenvironment. Developing drugs targeting these metabolic alterations could be a good direction. In this review, we briefly introduce metabolic changes/regulations of the tumor macroenvironment and microenvironment and summarize potential drugs targeting the metabolism in diffuse large B-cell lymphoma.
RESUMO
Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.
Assuntos
Fungos/genética , MicroRNAs/genética , Interferência de RNA , RNA Fúngico/genética , RNA Interferente Pequeno/genética , Fungos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Genoma , MicroRNAs/biossíntese , Modelos Genéticos , Neurospora crassa/genética , Neurospora crassa/metabolismo , RNA Fúngico/biossíntese , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The chloroplast genome of a dinoflagellate consists of a group of small circular DNA molecules (minicircles), most of which carry a single gene. With RT-PCR, primer extension, and Northern analyses, we show that the entire minicircle is transcribed and that some minicircles can produce RNAs larger than themselves. Using an RNA ligase-mediated rapid amplification of cDNA ends method, we were able to detect large processed precursors that are generated by endonucleolytic cleavage of an even longer molecule. This cleavage produces the mature mRNA 3'-end and at the same time the 5'-end of the precursor. The tRNAs encoded by the petD and psbE minicircles appear to be processed in the same way. We propose a "rolling circle" model for chloroplast transcription in which transcription would proceed continuously around the minicircular DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs.
Assuntos
DNA de Protozoário/metabolismo , Dinoflagellida/metabolismo , Genoma de Cloroplastos/fisiologia , Transcrição Gênica/fisiologia , DNA de Protozoário/genética , Dinoflagellida/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , RNA de Transferência/biossíntese , RNA de Transferência/genéticaRESUMO
Codon usage biases are found in all genomes and influence protein expression levels. The codon usage effect on protein expression was thought to be mainly due to its impact on translation. Here, we show that transcription termination is an important driving force for codon usage bias in eukaryotes. Using Neurospora crassa as a model organism, we demonstrated that introduction of rare codons results in premature transcription termination (PTT) within open reading frames and abolishment of full-length mRNA. PTT is a wide-spread phenomenon in Neurospora, and there is a strong negative correlation between codon usage bias and PTT events. Rare codons lead to the formation of putative poly(A) signals and PTT. A similar role for codon usage bias was also observed in mouse cells. Together, these results suggest that codon usage biases co-evolve with the transcription termination machinery to suppress premature termination of transcription and thus allow for optimal gene expression.
Assuntos
Códon/genética , Neurospora crassa/genética , Poliadenilação/genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Molecular , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Modelos Genéticos , Fases de Leitura Aberta/genéticaRESUMO
The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.
Assuntos
DNA de Cinetoplasto/genética , Dinoflagellida/genética , Plastídeos/genética , RNA de Transferência/genética , Transcrição Gênica , Animais , Sequência de Bases , Sequência Conservada , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.
Assuntos
RNA Polimerases Dirigidas por DNA/fisiologia , Dinoflagellida/genética , Genes Mitocondriais , RNA Nuclear/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sequência Conservada , Dinoflagellida/ultraestrutura , Genoma Mitocondrial , Filogenia , Subunidades ProteicasRESUMO
Substitutional editing increases genomic plasticity by changing or modifying bases at the RNA level. In this study we sequenced 10 mature chloroplast mRNAs, the chloroplast 16S rRNA and a partial chloroplast 23S rRNA from the dinoflagellate Heterocapsa triquetra, and found multiple types of substitutional editing, with A-to-G editing predominating. A-to-G editing of mRNAs converts two unusual AUA start codons into conventional AUG start codons, but three AUA start codons are not edited, showing that this dinoflagellate chloroplast has three possible start codons: AUG, AUA and UUG. To analyze the editing effects on rRNAs, we computationally predicted the secondary structure of the 16S rRNA based on the E. coli model. There are twenty editing sites in well-conserved regions of the secondary structure and eleven out of them restore conservation with other models. Moreover, A-to-G editing sites are frequently found in loop regions rather than double-stranded regions, suggesting that the A-to-G editing mechanism in dinoflagellate chloroplasts is different from that responsible for animal nuclear A-to-I(G) editing. The model of the edited 16S rRNA derived by the comparative method shares conserved secondary structural elements with other 16S rRNAs in spite of its very divergent primary sequence, supporting its role as a functional component of the chloroplast ribosome.