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1.
BMC Bioinformatics ; 20(1): 558, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703556

RESUMO

BACKGROUND: Vast amounts of next generation sequencing RNA data has been deposited in archives, accompanying very diverse original studies. The data is readily available also for other purposes such as genome annotation or transcriptome assembly. However, selecting a subset of available experiments, sequencing runs and reads for this purpose is a nontrivial task and complicated by the inhomogeneity of the data. RESULTS: This article presents the software VARUS that selects, downloads and aligns reads from NCBI's Sequence Read Archive, given only the species' binomial name and genome. VARUS automatically chooses runs from among all archived runs to randomly select subsets of reads. The objective of its online algorithm is to cover a large number of transcripts adequately when network bandwidth and computing resources are limited. For most tested species VARUS achieved both a higher sensitivity and specificity with a lower number of downloaded reads than when runs were manually selected. At the example of twelve eukaryotic genomes, we show that RNA-Seq that was sampled with VARUS is well-suited for fully-automatic genome annotation with BRAKER. CONCLUSIONS: With VARUS, genome annotation can be automatized to the extent that not even the selection and quality control of RNA-Seq has to be done manually. This introduces the possibility to have fully automatized genome annotation loops over potentially many species without incurring a loss of accuracy over a manually supervised annotation process.


Assuntos
Bases de Dados Genéticas , RNA Complementar/genética , Análise de Sequência de RNA/métodos , Software , Algoritmos , Animais , Drosophila melanogaster/genética , Eucariotos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Íntrons/genética , Anotação de Sequência Molecular , Transcriptoma/genética
2.
Genes Dev ; 33(21-22): 1555-1574, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31558568

RESUMO

The termination of pre-mRNA splicing functions to discard suboptimal substrates, thereby enhancing fidelity, and to release excised introns in a manner coupled to spliceosome disassembly, thereby allowing recycling. The mechanism of termination, including the RNA target of the DEAH-box ATPase Prp43p, remains ambiguous. We discovered a critical role for nucleotides at the 3' end of the catalytic U6 small nuclear RNA in splicing termination. Although conserved sequence at the 3' end is not required, 2' hydroxyls are, paralleling requirements for Prp43p biochemical activities. Although the 3' end of U6 is not required for recruiting Prp43p to the spliceosome, the 3' end cross-links directly to Prp43p in an RNA-dependent manner. Our data indicate a mechanism of splicing termination in which Prp43p translocates along U6 from the 3' end to disassemble the spliceosome and thereby release suboptimal substrates or excised introns. This mechanism reveals that the spliceosome becomes primed for termination at the same stage it becomes activated for catalysis, implying a requirement for stringent control of spliceosome activity within the cell.


Assuntos
Adenosina Trifosfatases/metabolismo , RNA Helicases DEAD-box/metabolismo , Processamento de RNA/fisiologia , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Íntrons/genética , Ligação Proteica , Processamento de RNA/genética
3.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514406

RESUMO

The quality of alfalfa, a main forage legume worldwide, is of great importance for the dairy industry and is affected by the content of triterpene saponins. These natural terpenoid products of triterpene aglycones are catalyzed by squalene synthase (SQS), a highly conserved enzyme present in eukaryotes. However, there is scare information on alfalfa SQS. Here, an open reading frame (ORF) of SQS was cloned from alfalfa. Sequence analysis showed MsSQS had the same exon/intron composition and shared high homology with its orthologs. Bioinformatic analysis revealed the deduced MsSQS had two transmembrane domains. When transiently expressed, GFP-MsSQS fusion protein was localized on the plasma membrane of onion epidermal cells. Removal of the C-terminal transmembrane domain of MsSQS improved solubility in Escherichia coli. MsSQS was preferably expressed in roots, followed by leaves and stems. MeJA treatment induced MsSQS expression and increased the content of total saponins. Overexpression of MsSQS in alfalfa led to the accumulation of total saponins, suggesting a correlation between MsSQS expression level with saponins content. Therefore, MsSQS is a canonical squalene synthase and contributes to saponin synthesis in alfalfa. This study provides a key candidate gene for genetic manipulation of the synthesis of triterpene saponins, which impact both plant and animal health.


Assuntos
Farnesil-Difosfato Farnesiltransferase/genética , Genes de Plantas , Medicago sativa/enzimologia , Medicago sativa/genética , Acetatos/farmacologia , Sequência de Aminoácidos , Membrana Celular/metabolismo , Clonagem Molecular , Ciclopentanos/farmacologia , Escherichia coli/metabolismo , Éxons/genética , Farnesil-Difosfato Farnesiltransferase/química , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Íntrons/genética , Cebolas/citologia , Oxilipinas/farmacologia , Filogenia , Epiderme Vegetal/citologia , Plantas Geneticamente Modificadas , Domínios Proteicos , Estrutura Secundária de Proteína , Saponinas/metabolismo , Solubilidade
4.
Biochemistry (Mosc) ; 84(8): 870-883, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522669

RESUMO

It has been suggested that RNA polymerase ribozyme displaying reverse transcriptase and integrase activities has played a vital role in the origin of life on Earth. Here, we present a hypothesis that formation of universal ancestral units of all living organisms - retroelements - in the evolution was mediated by reverse transcriptase. The propensity of retroelements to mutations and their insertion capacity have formed a basis for the origin of complex DNA structures - primary genomes - that have given rise to archaea, eukaryotes, bacteria, and viruses. Conserved properties of retroelements have been preserved throughout the evolution; their modifications have facilitated the emergence of mechanisms for the interactions between proteins and nucleic acids. Life has evolved due to insertional mutagenesis and competition of autonomously replicating polynucleotides that allowed to preserve structures with adaptive properties. We hypothesize that natural selection of mechanisms for the defense against insertions based on the ribonuclease activity of reverse transcriptase ribozyme has led to the emergence of all universal enzymatic systems for the processing of RNA molecules. These systems have been and still remain the key sources of structural and functional transformations of genomes in the course of evolution. The data presented in this review suggest that the process of translation, which unifies the nucleic acid and protein worlds, has developed as a modification of the defense mechanisms against insertions. Polypeptides formed by this defense system have potentiated the activity of ribozymes in the composition of ribonucleoproteins (RNPs) and even functionally replaced them as more efficient catalysts of biological reactions. Here, we analyze the mechanisms of retroelement involvement in the structural and regulatory transformations of eukaryotic genomes supposedly reflecting the adaptive principles that had originated during the beginning of life on Earth. Simultaneously with the evolution of existing proteins, retroelements have served as sources of new ribozymes, such as long non-coding RNAs. These ribozymes can function in complexes with proteins in the composition of RNPs, as well as display independent catalytic and translational activities; their genes have a potential for the transformation into protein-coding genes. Hence, the conserved principles of RNA, DNA, and proteins interregulation formed at the time of life origin on Earth have been used throughout the evolution.


Assuntos
Evolução Molecular , Origem da Vida , DNA Polimerase Dirigida por RNA/fisiologia , Animais , Elementos de DNA Transponíveis/genética , RNA Polimerases Dirigidas por DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Eucariotos/genética , Humanos , Íntrons/genética , Biossíntese de Proteínas , RNA Catalítico/fisiologia , RNA Longo não Codificante/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Retroelementos
5.
Nucleic Acids Res ; 47(19): 10327-10339, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31504764

RESUMO

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Sequência de Bases/genética , Domínio Catalítico/genética , Humanos , Íntrons/genética , Mutação/genética , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Dedos de Zinco/genética
6.
Plant Sci ; 287: 110203, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481208

RESUMO

Embryo and endosperm originate from the double fertilization, but they have different developmental fates and biological functions. We identified a previously undescribed maize seed mutant, wherein the embryo appears to be more severely affected than the endosperm (embryo-specific, emb). In the W22 background, the emb embryo arrests at the transition stage whereas its endosperm appears nearly normal in size. At maturity, the embryo in W22-emb is apparently small or even invisible. In contrast, the emb endosperm develops into a relative normal size. We cloned the mutant gene on the Chromosome 7L and designated it emb-7L. This gene is generally expressed, but it has a relatively higher expression level in leaves. Emb-7L encodes a chloroplast-localized P-type pentatricopeptide repeat (PPR) protein, consistent with the severe chloroplast deficiency in emb-7L albino seedling leaves. Full transcriptome analysis of the leaves of WT and emb-7L seedlings reveals that transcription of chloroplast protein-encoding genes are dramatically variable with pre-mRNA intron splicing apparently affected in a tissue-dependent pattern and the chloroplast structure and activity were dramatically affected including chloroplast membrane and photosynthesis machinery component and synthesis of metabolic products (e.g., fatty acids, amino acids, starch).


Assuntos
Proteínas de Plantas/genética , Processamento de RNA , Transcriptoma , Zea mays/genética , Cloroplastos/genética , Cloroplastos/ultraestrutura , Endosperma/embriologia , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos/genética , Íntrons/genética , Mutação , Fotossíntese , Folhas de Planta/embriologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Precursores de RNA/genética , Plântula/embriologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/ultraestrutura , Zea mays/embriologia , Zea mays/crescimento & desenvolvimento , Zea mays/ultraestrutura
7.
Int J Mol Sci ; 20(16)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434218

RESUMO

Seed number per capsule (SNC) is a major factor influencing seed yield and is an important trait with complex gene interaction effects. We first performed genetic analysis, gene cloning, and molecular mechanism study for an EMS-induced sesame mutant cs1 with fewer SNC and shorter capsule length (CL). The mutant traits were due to the pleiotropism of a regressive gene (Sics1). Capsule hormone determination showed that five out of 12 hormones, including auxin indole-3-acetic acid (IAA), had significantly different levels between wild type (WT) and mutant type (MT). KEGG pathway analysis showed that plant hormone signal transduction, especially the auxin signal transduction pathway, was the most abundant differentially expressed signaling pathway. After the cross-population association and regional genome screening, we found that three homozygous loci were retained in cs1. Further analysis of these three loci resulted in the identification of SiCRC as the candidate gene for cs1. SiCRC consists of seven exons and six introns encoding 163 amino acids. The SiCRC in cs1 showed a point mutation at intron 5 and exon 6 junction, resulting in the splice site being frame-shifted eight nucleotides further downstream, causing incorrect splicing. Taken together, we assumed the SNP mutation in SiCRC disrupted the function of the transcription factor, which might act downstream of the CRC-auxin signal transduction pathway, resulting in a shorter CL and less SNC mutation of cs1 in sesame. Our results highlight the molecular framework underlying the transcription factor CRC-mediated role of auxin transduction in SNC and CL development.


Assuntos
Proteínas de Plantas/metabolismo , Sementes/metabolismo , Sesamum/metabolismo , Éxons/genética , Regulação da Expressão Gênica de Plantas/genética , Pleiotropia Genética/genética , Pleiotropia Genética/fisiologia , Ácidos Indolacéticos/metabolismo , Íntrons/genética , Mutação , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Sementes/genética , Sesamum/genética
8.
PLoS Genet ; 15(8): e1008249, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437148

RESUMO

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function.


Assuntos
Processamento Alternativo , Evolução Molecular , Genoma Fúngico , Íntrons/genética , Saccharomyces cerevisiae/genética , RNA não Traduzido/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo
9.
BMC Genomics ; 20(1): 666, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438842

RESUMO

BACKGROUND: The class III peroxidase (PRX) gene family is a plant-specific member of the PRX superfamily that is closely related to various physiological processes, such as cell wall loosening, lignification, and abiotic and biotic stress responses. However, its classification, evolutionary history and gene expression patterns are unclear in wheat and Aegilops tauschii. RESULTS: Here, we identified 374, 159 and 169 PRXs in Triticum aestivum, Triticum urartu and Ae. tauschii, respectively. Together with PRXs detected from eight other plants, they were classified into 18 subfamilies. Among subfamilies V to XVIII, a conserved exon-intron structure within the "001" exon phases was detected in the PRX domain. Based on the analysis, we proposed a phylogenetic model to infer the evolutionary history of the exon-intron structures of PRX subfamilies. A comparative genomics analysis showed that subfamily VII could be the ancient subfamily that originated from green algae (Chlamydomonas reinhardtii). Further integrated analysis of chromosome locations and collinearity events of PRX genes suggested that both whole genome duplication (WGD) and tandem duplication (TD) events contributed to the expansion of T. aestivum PRXs (TaePRXs) during wheat evolution. To validate functions of these genes in the regulation of various physiological processes, the expression patterns of PRXs in different tissues and under various stresses were studied using public microarray datasets. The results suggested that there were distinct expression patterns among different tissues and PRXs could be involved in biotic and abiotic responses in wheat. qRT-PCR was performed on samples exposed to drought, phytohormone treatments and Fusarium graminearum infection to validate the microarray predictions. The predicted subcellular localizations of some TaePRXs were consistent with the confocal microscopy results. We predicted that some TaePRXs had hormone-responsive cis-elements in their promoter regions and validated these predicted cis-acting elements by sequencing promoters. CONCLUSION: In this study, identification, classification, evolution, and expression patterns of PRXs in wheat and relative plants were performed. Our results will provide information for further studies on the evolution and molecular mechanisms of wheat PRXs.


Assuntos
Aegilops/fisiologia , Evolução Molecular , Genômica , Peroxidase/genética , Estresse Fisiológico/genética , Triticum/fisiologia , Aegilops/enzimologia , Aegilops/genética , Éxons/genética , Genoma de Planta/genética , Íntrons/genética , Transcriptoma , Triticum/enzimologia , Triticum/genética
10.
BMC Evol Biol ; 19(1): 162, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375061

RESUMO

BACKGROUND: Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS: We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS: Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.


Assuntos
Sequência Conservada , Diplomonadida/genética , Íntrons/genética , Parabasalídeos/genética , Filogenia , Spliceossomos/genética , Regiões 5' não Traduzidas/genética , Pareamento de Bases/genética , Sequência de Bases , Genoma , Conformação de Ácido Nucleico , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Proteínas Ribossômicas/genética
11.
BMC Bioinformatics ; 20(1): 405, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345161

RESUMO

BACKGROUND: Next-generation sequencing technologies can produce tens of millions of reads, often paired-end, from transcripts or genomes. But few programs can align RNA on the genome and accurately discover introns, especially with long reads. We introduce Magic-BLAST, a new aligner based on ideas from the Magic pipeline. RESULTS: Magic-BLAST uses innovative techniques that include the optimization of a spliced alignment score and selective masking during seed selection. We evaluate the performance of Magic-BLAST to accurately map short or long sequences and its ability to discover introns on real RNA-seq data sets from PacBio, Roche and Illumina runs, and on six benchmarks, and compare it to other popular aligners. Additionally, we look at alignments of human idealized RefSeq mRNA sequences perfectly matching the genome. CONCLUSIONS: We show that Magic-BLAST is the best at intron discovery over a wide range of conditions and the best at mapping reads longer than 250 bases, from any platform. It is versatile and robust to high levels of mismatches or extreme base composition, and reasonably fast. It can align reads to a BLAST database or a FASTA file. It can accept a FASTQ file as input or automatically retrieve an accession from the SRA repository at the NCBI.


Assuntos
RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA/métodos , Software , Algoritmos , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Humanos , Íntrons/genética , Curva ROC , Fatores de Tempo
12.
Biomed Res Int ; 2019: 8620878, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31321242

RESUMO

CCN gene family members have recently been identified as multifunctional regulators involved in diverse biological functions, especially in vascular and skeletal development. In the present study, a comparative genomic and phylogenetic analysis was performed to show the similarities and differences in structure and function of CCNs from different organisms and to reveal their potential evolutionary relationship. First, CCN homologs of metazoans from different species were identified. Then we made multiple sequence alignments, MEME analysis, and functional sites prediction, which show the highly conserved structural features among CCN metazoans. The phylogenetic tree was further established, and thus CCNs were found undergoing extensive lineage-specific duplication events and lineage-specific expansion during the evolutionary process. Besides, comparative analysis about the genomic organization and chromosomal CCN gene surrounding indicated a clear orthologous relationship among these species counterparts. At last, based on these research results above, a potential evolutionary scenario was generated to overview the origin and evolution of the CCN gene family.


Assuntos
Proteínas de Sinalização Intercelular CCN/genética , Evolução Molecular , Genômica , Filogenia , Sequência de Aminoácidos/genética , Animais , Proteínas de Sinalização Intercelular CCN/classificação , Duplicação Gênica/genética , Íntrons/genética , Família Multigênica , Alinhamento de Sequência
13.
BMC Plant Biol ; 19(1): 307, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299897

RESUMO

BACKGROUND: DNA methylation is a crucial epigenetic modification, which is involved in many biological processes, including gene expression regulation, embryonic development, cell differentiation and genomic imprinting etc. And it also involves many key regulatory genes in eukaryotes. By tracing the evolutionary history of methylation-related genes, we can understand the origin and expansion time of these genes, which helps to understand the evolutionary history of plants, and we can also understand the changes of DNA methylation patterns in different species. However, most studies on the evolution of methylation-related genes failed to be carried out for the whole DNA methylation pathway. RESULTS: In this study, we conducted a comprehensive identification of 33 methylation-related genes in 77 species, and investigated gene origin and evolution throughout the plant kingdom. We found that the origin of genes responsible for methylation maintenance and demethylation evolved early, while most de novo methylation-related genes appeared late. The methylation-related genes were expanded by whole genome duplication and tandem replication, but were also accompanied by a large number of gene absence events in different species. The gene length and intron length varied a lot in different species, but exon structure and functional domains were relatively conserved. The phylogenetic relationships of methylation-related genes were traced to reveal the evolution history of DNA methylation in different species. The expression patterns of methylation-related genes have changed during the evolution of species, and the expression patterns of these genes in different species can be clustered into four categories. CONCLUSIONS: The study describes a global characterization of DNA methylation-related genes in the plant kingdom. The similarities and differences in origin time, gene structure and phylogenetic relationship of these genes lead us to understand the evolutionary conservation and dynamics of DNA methylation in plants.


Assuntos
Metilação de DNA , Epigênese Genética , Impressão Genômica , Plantas/genética , Evolução Molecular , Éxons/genética , Íntrons/genética , Filogenia
14.
Mol Biol (Mosk) ; 53(3): 411-420, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184606

RESUMO

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.


Assuntos
Processamento Alternativo/genética , Antitrombina III/genética , Sítios de Splice de RNA/genética , Éxons/genética , Humanos , Íntrons/genética
15.
Nucleic Acids Res ; 47(15): 8239-8254, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31216022

RESUMO

XAB2 is a multi-functional protein participating processes including transcription, splicing, DNA repair and mRNA export. Here, we report POLR2A, the largest catalytic subunit of RNA polymerase II, as a major target gene down-regulated after XAB2 depletion. XAB2 depletion led to severe splicing defects of POLR2A with significant intron retention. Such defects resulted in substantial loss of POLR2A at RNA and protein levels, which further impaired global transcription. Treatment of splicing inhibitor madrasin induced similar reduction of POLR2A. Screen using TMT-based quantitative proteomics identified several proteins involved in mRNA surveillance including Dom34 with elevated expression. Inhibition of translation or depletion of Dom34 rescued the expression of POLR2A by stabilizing its mRNA. Immuno-precipitation further confirmed that XAB2 associated with spliceosome components important to POLR2A expression. Domain mapping revealed that TPR motifs 2-4 and 11 of XAB2 were critical for POLR2A expression by interacting with SNW1. Finally, we showed POLR2A mediated cell senescence caused by XAB2 deficiency. Depletion of XAB2 or POLR2A induced cell senescence by up-regulation of p53 and p21, re-expression of POLR2A after XAB2 depletion alleviated cellular senescence. These data together support that XAB2 serves as a guardian of POLR2A expression to ensure global gene expression and antagonize cell senescence.


Assuntos
Senescência Celular/genética , RNA Polimerases Dirigidas por DNA/genética , Íntrons/genética , Fatores de Transcrição/genética , Transcrição Genética , Linhagem Celular , Linhagem Celular Tumoral , RNA Polimerases Dirigidas por DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
16.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253655

RESUMO

Dietary restriction (DR) increases life span through adaptive changes in gene expression. To understand more about these changes, we analyzed the transcriptome and translatome of Caenorhabditis elegans subjected to DR. Transcription of muscle regulatory and structural genes increased, whereas increased expression of amino acid metabolism and neuropeptide signaling genes was controlled at the level of translation. Evaluation of posttranscriptional regulation identified putative roles for RNA-binding proteins, RNA editing, miRNA, alternative splicing, and nonsense-mediated decay in response to nutrient limitation. Using RNA interference, we discovered several differentially expressed genes that regulate life span. We also found a compensatory role for translational regulation, which offsets dampened expression of a large subset of transcriptionally down-regulated genes. Furthermore, 3' UTR editing and intron retention increase under DR and correlate with diminished translation, whereas trans-spliced genes are refractory to reduced translation efficiency compared with messages with the native 5' UTR. Finally, we find that smg-6 and smg-7, which are genes governing selection and turnover of nonsense-mediated decay targets, are required for increased life span under DR.


Assuntos
Caenorhabditis elegans/genética , Restrição Calórica , Longevidade/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas , Processamento Alternativo , Aminoácidos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Íntrons/genética , MicroRNAs/metabolismo , Proteínas Musculares/metabolismo , Neuropeptídeos/metabolismo , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Motivos de Ligação ao RNA , Telomerase/metabolismo , Transcriptoma
17.
Mol Cell ; 75(2): 324-339.e11, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31155380

RESUMO

Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.


Assuntos
Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Códon sem Sentido/química , Éxons/genética , Humanos , Íntrons/genética , Mutação/genética , Estabilidade de RNA/genética , RNA Mensageiro/química , Imagem Individual de Molécula
18.
RNA ; 25(9): 1130-1149, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31175170

RESUMO

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.


Assuntos
Nanismo/genética , Retardo do Crescimento Fetal/genética , Microcefalia/genética , Osteocondrodisplasias/genética , Processamento de RNA/genética , Transcriptoma/genética , Adulto , Idoso , Sequência de Bases/genética , Pré-Escolar , Sequência Consenso/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Lactente , Íntrons/genética , Masculino , Pessoa de Meia-Idade , RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Spliceossomos/genética , Adulto Jovem
19.
Cells ; 8(6)2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146469

RESUMO

: The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrus bretschneideri, Prunus persica, Citrus sinensis, Vitis vinifera, and Prunus mume), and 10 of these sequences came from Pyrus bretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O2.-) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.


Assuntos
Simulação por Computador , Frutas/genética , Genes de Plantas , Lignina/metabolismo , Família Multigênica , NADPH Oxidases/genética , Pyrus/enzimologia , Pyrus/genética , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Sequência Conservada/genética , Éxons/genética , Frutas/efeitos dos fármacos , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Tamanho do Genoma , Íntrons/genética , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Motivos de Nucleotídeos/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Filogenia , Reguladores de Crescimento de Planta/farmacologia , Regiões Promotoras Genéticas/genética , Pyrus/efeitos dos fármacos , Sintenia/genética , Árvores/enzimologia , Árvores/genética
20.
Cells ; 8(6)2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234415

RESUMO

Single nucleotide polymorphisms located in 5' untranslated regions (5'UTRs) can regulate gene expression and have clinical impact. Recognition of functionally significant sequences within 5'UTRs is crucial in next-generation sequencing applications. Furthermore, information about the behavior of 5'UTRs during gene evolution is scarce. Using the example of the ATP-binding cassette transporter A1 (ABCA1) gene (Tangier disease), we describe our algorithm for functionally significant sequence finding. 5'UTR features (upstream start and stop codons, open reading frames (ORFs), GC content, motifs, and secondary structures) were studied using freely available bioinformatics tools in 55 vertebrate orthologous genes obtained from Ensembl and UCSC. The most conserved sequences were suggested as hot spots. Exon and intron enhancers and silencers (sc35, ighg2 cgamma2, ctnt, gh-1, and fibronectin eda exon), transcription factors (TFIIA, TATA, NFAT1, NFAT4, and HOXA13), some of them cancer related, and microRNA (hsa-miR-4474-3p) were localized to these regions. An upstream ORF, overlapping with the main ORF in primates and possibly coding for a small bioactive peptide, was also detected. Moreover, we showed several features of 5'UTRs, such as GC content variation, hairpin structure conservation or 5'UTR segmentation, which are interesting from a phylogenetic point of view and can stimulate further evolutionary oriented research.


Assuntos
Regiões 5' não Traduzidas/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Vertebrados/genética , Transportador 1 de Cassete de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Composição de Bases/genética , Sequência de Bases , Sequência Conservada/genética , Elementos Facilitadores Genéticos/genética , Humanos , Íntrons/genética , Mamíferos/genética , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Fases de Leitura Aberta/genética , Filogenia , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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