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2.
Genome Biol ; 23(1): 149, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35799267

RESUMEN

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.


Asunto(s)
Arabidopsis , Transcriptoma , Empalme Alternativo , Arabidopsis/genética , Perfilación de la Expresión Génica/métodos , RNA-Seq , Análisis de Secuencia de ARN/métodos
3.
Cell Rep ; 36(10): 109676, 2021 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-34496244

RESUMEN

For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Light-triggered chloroplast retrograde signals control alternative splicing in Arabidopsis thaliana. Here, we provide evidence that light regulates the expression of a core set of splicing-related factors in roots. Alternative splicing responses in roots are not directly caused by light but are instead most likely triggered by photosynthesized sugars. The target of rapamycin (TOR) kinase plays a key role in this shoot-to-root signaling pathway. Knocking down TOR expression or pharmacologically inhibiting TOR activity disrupts the alternative splicing responses to light and exogenous sugars in roots. Consistently, splicing decisions are modulated by mitochondrial activity in roots. In conclusion, by activating the TOR pathway, sugars act as mobile signals to coordinate alternative splicing responses to light throughout the whole plant.


Asunto(s)
Empalme Alternativo/genética , Luz , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas , Sirolimus/metabolismo
4.
Nucleic Acids Res ; 49(2): 1133-1151, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406240

RESUMEN

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.


Asunto(s)
Empalme Alternativo/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Técnicas de Silenciamiento del Gen , Degradación de ARNm Mediada por Codón sin Sentido , Isoformas de Proteínas/genética , Interferencia de ARN , Precursores del ARN/metabolismo , ARN de Planta/metabolismo , Proteínas de Arabidopsis/biosíntesis , Exones , Genes de Plantas , Células HeLa , Humanos , MicroARNs/genética , Plantas Modificadas Genéticamente , Isoformas de Proteínas/biosíntesis , Protoplastos/metabolismo , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , ARN de Planta/genética , Factores de Empalme Serina-Arginina/biosíntesis , Factores de Empalme Serina-Arginina/genética , Transcripción Genética , Transfección
5.
Plant J ; 94(6): 1010-1022, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29602264

RESUMEN

The ability to adapt growth and development to temperature variations is crucial to generate plant varieties resilient to predicted temperature changes. However, the mechanisms underlying plant response to progressive increases in temperature have just started to be elucidated. Here, we report that the cyclin-dependent kinase G1 (CDKG1) is a central element in a thermo-sensitive mRNA splicing cascade that transduces changes in ambient temperature into differential expression of the fundamental spliceosome component, ATU2AF65A. CDKG1 is alternatively spliced in a temperature-dependent manner. We found that this process is partly dependent on both the cyclin-dependent kinase G2 (CDKG2) and the interacting co-factor CYCLIN L1 (CYCL1), resulting in two distinct messenger RNAs. The relative abundance of both CDKG1 transcripts correlates with ambient temperature and possibly with different expression levels of the associated protein isoforms. Both CDKG1 alternative transcripts are necessary to fully complement the expression of ATU2AF65A across the temperature range. Our data support a previously unidentified temperature-dependent mechanism based on the alternative splicing (AS) of CDKG1 and regulated by CDKG2 and CYCL1. We propose that changes in ambient temperature affect the relative abundance of CDKG1 transcripts, and this in turn translates into differential CDKG1 protein expression coordinating the AS of ATU2AF65A.


Asunto(s)
Empalme Alternativo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Factores de Empalme de ARN/metabolismo , Empalme Alternativo/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fraccionamiento Celular , Regulación de la Expresión Génica de las Plantas/genética , Factores de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Empalmosomas/metabolismo , Temperatura
7.
Nucleic Acids Res ; 45(9): 5061-5073, 2017 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-28402429

RESUMEN

Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.


Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Genes de Insecto , Transcriptoma , Variación Genética , Proteómica , ARN no Traducido , Valores de Referencia , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Transcripción Genética
8.
Nucleic Acids Res ; 44(4): 1703-17, 2016 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-26682798

RESUMEN

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.


Asunto(s)
Empalme Alternativo/genética , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Precursores del ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , ADN/química , ADN/genética , Reparación del ADN/genética , ARN/química , ARN/genética , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Schizosaccharomyces/genética , Empalmosomas/genética , Empalmosomas/metabolismo
9.
Plant Cell ; 27(8): 2083-7, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26286536

RESUMEN

Transcript annotation in plant databases is incomplete and often inaccurate, leading to misinterpretation. As more and more RNA-seq data are generated, plant scientists need to be aware of potential pitfalls and understand the nature and impact of specific alternative splicing transcripts on protein production. A primary area of concern and the topic of this article is the (mis)annotation of open reading frames and premature termination codons. The basic message is that to adequately address expression and functions of transcript isoforms, it is necessary to be able to predict their fate in terms of whether protein isoforms are generated or specific transcripts are unproductive or degraded.


Asunto(s)
Empalme Alternativo , Proteínas de Plantas/genética , Plantas/genética , Biosíntesis de Proteínas/genética , Modelos Genéticos , Sistemas de Lectura Abierta/genética , Isoformas de Proteínas/genética , Estabilidad del ARN , ARN Mensajero/genética
10.
New Phytol ; 208(1): 96-101, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26111100

RESUMEN

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.


Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Isoformas de Proteínas/análisis , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Algoritmos , Secuencia de Bases , Conjuntos de Datos como Asunto , Genes de Plantas , Empalme del ARN , Valores de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas Informáticos , Transcriptoma
11.
Genome Res ; 25(7): 995-1007, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25934563

RESUMEN

Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events.


Asunto(s)
Empalme Alternativo , Exones , Intrones , Sistemas de Lectura Abierta , Proteómica , Arabidopsis/genética , Arabidopsis/metabolismo , Neoplasias de la Mama , Evolución Molecular , Femenino , Regulación de la Expresión Génica de las Plantas , Humanos , Especificidad de Órganos/genética , Biosíntesis de Proteínas , Transporte de ARN , Estrés Fisiológico/genética , Transcriptoma
12.
Plant Signal Behav ; 9(11): e976150, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25482785

RESUMEN

Plants rely on a sophisticated light sensing and signaling system that allows them to respond to environmental changes. Photosensory protein systems -phytochromes, cryptochromes, phototropins, and ultraviolet (UV)-B photoreceptors- have evolved to let plants monitor light conditions and regulate different levels of gene expression and developmental processes. However, even though photoreceptor proteins are best characterized and deeply studied, it is also known that chloroplasts are able to sense light conditions and communicate the variations to the nucleus that adjust its transcriptome to the changing environment. The redox state of components of the photosynthetic electron transport chain works as a sensor of photosynthetic activity and can affect nuclear gene expression by a retrograde signaling pathway. Recently, our groups showed that a retrograde signaling pathway can modulate the alternative splicing process, revealing a novel layer of gene expression control by chloroplast retrograde signaling.


Asunto(s)
Núcleo Celular/genética , Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Núcleo Celular/efectos de la radiación , Cloroplastos/efectos de la radiación , Fototransducción/genética , Fototransducción/efectos de la radiación , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/metabolismo
13.
Plant Methods ; 10: 15, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24991230

RESUMEN

BACKGROUND: A multitude of different imaging systems are already available to image genetically altered RNA species; however, only a few of these techniques are actually suitable to visualize endogenous RNA. One possibility is to use fluorescently-labelled and hybridization-sensitive probes. In order to yield more information about the exact localization and movement of a single RNA molecule, it is necessary to image such probes with highly sensitive microscope setups. More challenges arise if such experiments are conducted in plant cells due to their high autofluorescence and demanding transfection procedures. RESULTS: Here, we report in planta imaging of single RNA molecules using fluorescently labeled molecular beacons. We tested three different transfection protocols in order to identify optimal conditions for transfection of fluorescent DNA probes and their subsequent detection at the single molecule level. CONCLUSIONS: We found that an optimized heat shock protocol provided a vastly improved transfection method for small DNA molecules which were used for subsequent single RNA molecule detection in living plant suspension cells.

14.
Science ; 344(6182): 427-30, 2014 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-24763593

RESUMEN

Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action, we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.


Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Plastoquinona/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Relojes Circadianos , Dibromotimoquinona/farmacología , Diurona/farmacología , Transporte de Electrón/efectos de los fármacos , Luz , Modelos Biológicos , Oxidación-Reducción , Fotosíntesis/efectos de los fármacos , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Plantones/genética , Plantones/metabolismo , Transducción de Señal
15.
Plant Cell ; 26(2): 754-64, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24532591

RESUMEN

Alternative splicing (AS) is an important regulatory process that leads to the creation of multiple RNA transcripts from a single gene. Alternative transcripts often carry premature termination codons (PTCs), which trigger nonsense-mediated decay (NMD), a cytoplasmic RNA degradation pathway. However, intron retention, the most prevalent AS event in plants, often leads to PTC-carrying splice variants that are insensitive to NMD; this led us to question the fate of these special RNA variants. Here, we present an innovative approach to monitor and characterize endogenous mRNA splice variants within living plant cells. This method combines standard confocal laser scanning microscopy for molecular beacon detection with a robust statistical pipeline for sample comparison. We demonstrate this technique on the localization of NMD-insensitive splice variants of two Arabidopsis thaliana genes, RS2Z33 and the SEF factor. The experiments reveal that these intron-containing splice variants remain within the nucleus, which allows them to escape the NMD machinery. Moreover, fluorescence recovery after photobleaching experiments in the nucleoplasm show a decreased mobility of intron-retained mRNAs compared with fully spliced RNAs. In addition, differences in mobility were observed for an mRNA dependent on its origin from an intron-free or an intron-containing gene.


Asunto(s)
Empalme Alternativo/genética , Arabidopsis/genética , Núcleo Celular/metabolismo , Imagen Molecular/métodos , Degradación de ARNm Mediada por Codón sin Sentido/genética , Células Vegetales/metabolismo , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Supervivencia Celular , Electroporación , Recuperación de Fluorescencia tras Fotoblanqueo , Protoplastos/citología , Protoplastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección
16.
RNA Biol ; 11(10): 1215-20, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25590224

RESUMEN

Gene expression regulation relies on a variety of molecular mechanisms affecting different steps of a messenger RNA (mRNA) life: transcription, processing, splicing, alternative splicing, transport, translation, storage and decay. Light induces massive reprogramming of gene expression in plants. Differences in alternative splicing patterns in response to environmental stimuli suggest that alternative splicing plays an important role in plant adaptation to changing life conditions. In a recent publication, our laboratories showed that light regulates alternative splicing of a subset of Arabidopsis genes encoding proteins involved in RNA processing by chloroplast retrograde signals. The light effect on alternative splicing is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. These results point at alternative splicing regulation by retrograde signals as an important mechanism for plant adaptation to their environment.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Luz , Fotosíntesis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación
17.
Plant Cell ; 25(10): 3657-83, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24179125

RESUMEN

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.


Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica de las Plantas , Desarrollo de la Planta , Epigénesis Genética , Precursores del ARN/genética , ARN de Planta/genética , Transducción de Señal , Empalmosomas/metabolismo
18.
EMBO Rep ; 14(7): 622-8, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23681439

RESUMEN

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop-off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5'ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen-triggered accumulation of miR163 and proper regulation of at least one of its targets.


Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Intrones , MicroARNs/genética , Precursores del ARN/genética , Empalme Alternativo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , MicroARNs/biosíntesis , Mutación , Poli A/genética , Poli A/metabolismo , Pseudomonas syringae/fisiología , Precursores del ARN/biosíntesis
19.
Nucleic Acids Res ; 41(3): 1783-96, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23248006

RESUMEN

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ciclofilinas/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Genómica/métodos , Motivos de Nucleótidos , ARN Polimerasa II/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo
20.
Trends Plant Sci ; 17(10): 616-23, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22743067

RESUMEN

More than 60% of intron-containing genes undergo alternative splicing (AS) in plants. This number will increase when AS in different tissues, developmental stages, and environmental conditions are explored. Although the functional impact of AS on protein complexity is still understudied in plants, recent examples demonstrate its importance in regulating plant processes. AS also regulates transcript levels and the link with nonsense-mediated decay and generation of unproductive mRNAs illustrate the need for both transcriptional and AS data in gene expression analyses. AS has influenced the evolution of the complex networks of regulation of gene expression and variation in AS contributed to adaptation of plants to their environment and therefore will impact strategies for improving plant and crop phenotypes.


Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica de las Plantas , Plantas/metabolismo , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Relojes Circadianos , Epigénesis Genética , Evolución Molecular , Genes de Plantas , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Poliploidía , Estabilidad Proteica , ARN Mensajero/genética , ARN de Planta/genética , Transcripción Genética
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