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1.
Plant Cell ; 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38869231

RESUMEN

Decapping is a crucial step in mRNA degradation in eucaryotes and requires the formation of a holoenzyme complex between the decapping enzyme DECAPPING 2 (DCP2) and the decapping enhancer DCP1. In Arabidopsis (Arabidopsis thaliana), DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a direct protein partner of DCP1. The function of both DNE1 and decapping are necessary to maintain phyllotaxis, the regularity of organ emergence in the apex. In this study, we combined in vivo mRNA editing, RNA degradome sequencing, transcriptomics and small RNA-omics to identify targets of DNE1 and study how DNE1 and DCP2 cooperate in controlling mRNA fate. Our data reveal that DNE1 mainly contacts and cleaves mRNAs in the coding sequence and has sequence cleavage preferences. DNE1 targets are also degraded through decapping, and both RNA degradation pathways influence the production of mRNA-derived small interfering RNAs. Finally, we detected mRNA features enriched in DNE1 targets including RNA G-quadruplexes and translated upstream open reading frames. Combining these four complementary high-throughput sequencing strategies greatly expands the range of DNE1 targets and allowed us to build a conceptual framework describing the influence of DNE1 and decapping on mRNA fate. These data will be crucial to unveil the specificity of DNE1 action and understand its importance for developmental patterning.

2.
Nucleic Acids Res ; 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38721772

RESUMEN

Until recently, the general 5'-3' mRNA decay was placed in the cytosol after the mRNA was released from ribosomes. However, the discovery of an additional 5' to 3' pathway, the Co-Translational mRNA Decay (CTRD), changed this paradigm. Up to date, defining the real contribution of CTRD in the general mRNA turnover has been hardly possible as the enzyme involved in this pathway is also involved in cytosolic decay. Here we overcame this obstacle and created an Arabidopsis line specifically impaired for CTRD called XRN4ΔCTRD. Through a genome-wide analysis of mRNA decay rate in shoot and root, we tested the importance of CTRD in mRNA turnover. First, we observed that mRNAs tend to be more stable in root than in shoot. Next, using XRN4ΔCTRD line, we demonstrated that CTRD is a major determinant in mRNA turnover. In shoot, the absence of CTRD leads to the stabilization of thousands of transcripts while in root its absence is highly compensated resulting in faster decay rates. We demonstrated that this faster decay rate is partially due to the XRN4-dependent cytosolic decay. Finally, we correlated this organ-specific effect with XRN4ΔCTRD line phenotypes revealing a crucial role of CTRD in mRNA homeostasis and proper organ development.

3.
New Phytol ; 233(1): 251-264, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34643285

RESUMEN

Arabidopsis thaliana seed germination is marked by extensive translational control at two critical phase transitions. The first transition refers to the start of hydration, the hydration translational shift. The second shift, the germination translational shift (GTS) is the phase between testa rupture and radicle protrusion at which the seed makes the all or nothing decision to germinate. The mechanism behind the translational regulation at these phase transitions is unknown. RNA binding proteins (RBPs) are versatile players in the post-transcriptional control of messenger RNAs (mRNAs) and as such candidates for regulating translation during seed germination. Here, we report the mRNA binding protein repertoire of seeds during the GTS. Thirty seed specific RBPs and 22 dynamic RBPs were identified during the GTS, like the putative RBP Vacuolar ATPase subunit A and RBP HSP101. Several stress granule markers were identified in this study, which suggests that seeds are prepared to quickly adapt the translation of specific mRNAs in response to changes in environmental conditions during the GTS. Taken together this study provides a detailed insight into the world of RBPs during seed germination and their possible regulatory role during this developmentally regulated process.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Germinación , Proteoma , ARN Mensajero/genética , Semillas/genética , Gránulos de Estrés
4.
Plant Physiol ; 184(3): 1251-1262, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32913043

RESUMEN

RNA turnover is a general process that maintains appropriate mRNA abundance at the posttranscriptional level. Although long thought to be antagonistic to translation, discovery of the 5' to 3' cotranslational mRNA decay pathway demonstrated that both processes are intertwined. Cotranslational mRNA decay globally shapes the transcriptome in different organisms and in response to stress; however, the dynamics of this process during plant development is poorly understood. In this study, we used a multiomics approach to reveal the global landscape of cotranslational mRNA decay during Arabidopsis (Arabidopsis thaliana) seedling development. We demonstrated that cotranslational mRNA decay is regulated by developmental cues. Using the EXORIBONUCLEASE4 (XRN4) loss-of-function mutant, we showed that XRN4 poly(A+) mRNA targets are largely subject to cotranslational decay during plant development. As cotranslational mRNA decay is interconnected with translation, we also assessed its role in translation efficiency. We discovered that clusters of transcripts were specifically subjected to cotranslational decay in a developmental-dependent manner to modulate their translation efficiency. Our approach allowed the determination of a cotranslational decay efficiency that could be an alternative to other methods to assess transcript translation efficiency. Thus, our results demonstrate the prevalence of cotranslational mRNA decay in plant development and its role in translational control.


Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Biosíntesis de Proteínas/fisiología , Estabilidad del ARN/fisiología , ARN de Planta/fisiología , Variación Genética , Genotipo , Mutación , Estabilidad del ARN/genética , Plantones/genética , Plantones/crecimiento & desarrollo
5.
Plant Cell ; 30(5): 986-1005, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29618631

RESUMEN

Methylations at position N6 of internal adenosines (m6As) are the most abundant and widespread mRNA modifications. These modifications play crucial roles in reproduction, growth, and development by controlling gene expression patterns at the posttranscriptional level. Their function is decoded by readers that share the YTH domain, which forms a hydrophobic pocket that directly accommodates the m6A residues. While the physiological and molecular functions of YTH readers have been extensively studied in animals, little is known about plant readers, even though m6As are crucial for plant survival and development. Viridiplantae contains high numbers of YTH domain proteins. Here, we performed comprehensive evolutionary analysis of YTH domain proteins and demonstrated that they are highly likely to be actual readers with redundant as well as specific functions. We also show that the ECT2 protein from Arabidopsis thaliana binds to m6A-containing RNAs in vivo and that this property relies on the m6A binding pocket carried by its YTH domain. ECT2 is cytoplasmic and relocates to stress granules upon heat exposure, suggesting that it controls mRNA fate in the cytosol. Finally, we demonstrate that ECT2 acts to decode the m6A signal in the trichome and is required for their normal branching through controlling their ploidy levels.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Tricomas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Unión Proteica
6.
RNA Biol ; 17(8): 1137-1148, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-31994438

RESUMEN

Transfer RNA-derived fragments (tRFs) exist in all branches of life. They are involved in RNA degradation, regulation of gene expression, ribosome biogenesis. In archaebacteria, kinetoplastid, yeast, and human cells, they were also shown to regulate translation. In Arabidopsis, the tRFs population fluctuates under developmental or environmental conditions but their functions are yet poorly understood. Here, we show that populations of long (30-35 nt) or short (19-25 nt) tRFs produced from Arabidopsis tRNAs can inhibit in vitro translation of a reporter gene. Analysing a series of oligoribonucleotides mimicking natural tRFs, we demonstrate that only a limited set of tRFs possess the ability to affect protein synthesis. Out of a dozen of tRFs, only two deriving from tRNAAla(AGC) and tRNAAsn(GUU) strongly attenuate translation in vitro. Contrary to human tRF(Ala), the 4 Gs present at the 5' extremity of Arabidopsis tRF(Ala) are not implicated in this inhibition while the G18 and G19 residues are essential. Protein synthesis inhibition by tRFs does not require complementarity with the translated mRNA but, having the capability to be associated with polyribosomes, tRFs likely act as general modulation factors of the translation process in plants.


Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Biosíntesis de Proteínas , ARN de Transferencia/genética , ARN no Traducido/genética , Conformación de Ácido Nucleico , Polirribosomas/metabolismo , ARN de Transferencia/química , ARN no Traducido/química
7.
Plant Physiol ; 174(2): 1216-1225, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28381501

RESUMEN

Heat shock (HS) is known to have a profound impact on gene expression at different levels, such as inhibition of protein synthesis, in which HS blocks translation initiation and induces the sequestration of mRNAs into stress granules (SGs) or P-bodies for storage and/or decay. SGs prevent the degradation of the stored mRNAs, which can be reengaged into translation in the recovery period. However, little is known on the mRNAs stored during the stress, how these mRNAs are released from SGs afterward, and what the functional importance is of this process. In this work, we report that Arabidopsis HEAT SHOCK PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery and SG dissociation after HS Using RNA sequencing and RNA immunoprecipitation approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially stored during HS and that these mRNAs were released and translated in an HSP101-dependent manner during recovery. By 15N incorporation and polysome profile analyses, we observed that these released mRNAs contributed to the production of new ribosomes to enhance translation. We propose that, after HS, HSP101 is required for the efficient release of RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by producing new RPs.


Asunto(s)
Respuesta al Choque Térmico/genética , Proteínas de Plantas/metabolismo , Proteínas Ribosómicas/genética , Factores de Transcripción/metabolismo , Gránulos Citoplasmáticos/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Mutación/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Transcripción Genética
8.
Plant Cell ; 26(3): 1330-44, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24668745

RESUMEN

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.


Asunto(s)
Arabidopsis/genética , Cromatina/metabolismo , ADN Ribosómico/genética , Duplicación de Gen , Fosfoproteínas/genética , ARN Ribosómico/genética , Proteínas de Unión al ARN/genética , Variaciones en el Número de Copia de ADN , Metilación de ADN , Genes de Plantas , Regiones Promotoras Genéticas , Nucleolina
9.
Nucleic Acids Res ; 43(8): 4121-32, 2015 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-25845591

RESUMEN

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5'-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5'-ribosome pausing leading to the XRN4-mediated 5'-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of 'non-aberrant' mRNAs.


Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Calor , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico/genética , Arabidopsis/metabolismo , Regulación hacia Abajo , Exorribonucleasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Polirribosomas/metabolismo , Estabilidad del ARN
10.
RNA ; 19(1): 36-50, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23148093

RESUMEN

La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3' UTRs and led to their neofunctionalization.


Asunto(s)
Autoantígenos/química , Autoantígenos/clasificación , Evolución Molecular , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/clasificación , Ribonucleoproteínas/química , Ribonucleoproteínas/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Autoantígenos/genética , Secuencia Conservada/genética , Humanos , Modelos Genéticos , Modelos Moleculares , Filogenia , Proteínas de Unión a Poli(A)/genética , Ribonucleoproteínas/genética , Alineación de Secuencia , Antígeno SS-B
11.
Nucleic Acids Res ; 41(14): 7115-27, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23748567

RESUMEN

Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12-15 As. Deadenylation leads to decapping and to subsequent 5' to 3' degradation by XRN proteins, or alternatively 3' to 5' degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay.


Asunto(s)
Nucleótidos de Adenina/metabolismo , Proteínas de Arabidopsis/metabolismo , Oligorribonucleótidos/metabolismo , Procesamiento de Término de ARN 3' , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/metabolismo , Uridina Monofosfato/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Mutación , Polirribosomas/metabolismo , ARN Nucleotidiltransferasas/genética , Estabilidad del ARN , Uridina/metabolismo
12.
Genome Biol Evol ; 15(6)2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37220646

RESUMEN

Although duplications have long been recognized as a fundamental process driving major evolutionary innovations, direct estimates of spontaneous chromosome duplication rates, leading to aneuploid karyotypes, are scarce. Here, from mutation accumulation (MA) experiments, we provide the first estimates of spontaneous chromosome duplication rates in six unicellular eukaryotic species, which range from 1 × 10-4 to 1 × 10-3 per genome per generation. Although this is ∼5 to ∼60 times less frequent than spontaneous point mutations per genome, chromosome duplication events can affect 1-7% of the total genome size. In duplicated chromosomes, mRNA levels reflected gene copy numbers, but the level of translation estimated by polysome profiling revealed that dosage compensation must be occurring. In particular, one duplicated chromosome showed a 2.1-fold increase of mRNA but translation rates were decreased to 0.7-fold. Altogether, our results support previous observations of chromosome-dependent dosage compensation effects, providing evidence that compensation occurs during translation. We hypothesize that an unknown posttranscriptional mechanism modulates the translation of hundreds of transcripts from genes located on duplicated regions in eukaryotes.


Asunto(s)
Duplicación Cromosómica , Genoma , Humanos , Dosificación de Gen , Cromosomas/genética , ARN Mensajero/genética , Duplicación de Gen
13.
Elife ; 122023 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-37773033

RESUMEN

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Lignina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Procesamiento Proteico-Postraduccional , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas
14.
Nat Commun ; 14(1): 254, 2023 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-36650156

RESUMEN

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.


Asunto(s)
Agroquímicos , Control de Malezas , Agroquímicos/farmacología , Resistencia a los Herbicidas/genética , Malezas/genética , Péptidos , Productos Agrícolas/genética
15.
Plants (Basel) ; 11(19)2022 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-36235485

RESUMEN

mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.

16.
Cell Rep ; 41(11): 111784, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36516773

RESUMEN

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.


Asunto(s)
Cromatina , Respuesta al Choque Térmico , Humanos , Respuesta al Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Expresión Génica , Factores de Empalme de ARN/metabolismo , Proteínas del Tejido Nervioso/metabolismo
17.
BMC Biol ; 8: 18, 2010 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-20202192

RESUMEN

BACKGROUND: Technological advances have enabled the accurate quantification of gene expression, even within single cell types. While transcriptome analyses are routinely performed, most experimental designs only provide snapshots of gene expression. Molecular mechanisms underlying cell fate or positional signalling have been revealed through these discontinuous datasets. However, in developing multicellular structures, temporal and spatial cues, known to directly influence transcriptional networks, get entangled as the cells are displaced and expand. Access to an unbiased view of the spatiotemporal regulation of gene expression occurring during development requires a specific framework that properly quantifies the rate of change of a property in a moving and expanding element, such as a cell or an organ segment. RESULTS: We show how the rate of change in gene expression can be quantified by combining kinematics and real-time polymerase chain reaction data in a mechanistic model which considers any organ as a continuum. This framework was applied in order to assess the developmental regulation of the two reference genes Actin11 and Elongation Factor 1-beta in the apex of poplar root. The growth field was determined by time-lapse photography and transcript density was obtained at high spatial resolution. The net accumulation rates of the transcripts of the two genes were found to display highly contrasted developmental profiles. Actin11 showed pulses of up and down regulation in the accelerating and decelerating parts of the growth zone while the dynamic of EF1beta were much slower. This framework provides key information about gene regulation in a developing organ, such as the location, the duration and the intensity of gene induction/repression. CONCLUSIONS: We demonstrated that gene expression patterns can be monitored using the continuity equation without using mutants or reporter constructions. Given the rise of imaging technologies, this framework in our view opens a new way to dissect the molecular basis of growth regulation, even in non-model species or complex structures.


Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Raíces de Plantas/genética , Populus/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Modelos Teóricos , Factor 1 de Elongación Peptídica/genética , Reacción en Cadena de la Polimerasa
18.
Plants (Basel) ; 10(3)2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33804539

RESUMEN

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5'monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5'P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5'P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

19.
Nat Plants ; 7(8): 1050-1064, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34373603

RESUMEN

Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.


Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Fosfatos/metabolismo , Células Vegetales/metabolismo , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Transcripción Genética , Regulación de la Expresión Génica de las Plantas , Cinética , ARN Polimerasa II/genética
20.
FEBS Lett ; 581(27): 5295-99, 2007 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-18028913

RESUMEN

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is unique in the first part of the cytoplasmic isoprenoid pathway, as it contains a membrane domain that includes ER-specific retention motifs. When fused to GFP, this domain targets two tobacco BY-2 HMGR isoforms differentially. While the first isoform is ER-localized, a second stress-induced one forms globular structures connected by tubular structures. A serine positioned upstream of the ER retention motif seems to be implicated in this specific subcellular localization. Surprisingly, these structures are closely connected to F-actin, and their intactness is dependent upon the integrity of the filaments or the action of a calmodulin antagonist.


Asunto(s)
Actinas/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Nicotiana/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN de Plantas/genética , Retículo Endoplásmico/enzimología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Fracciones Subcelulares/enzimología , Nicotiana/genética
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