RESUMEN
MicroRNAs (miRNAs) are produced from highly structured primary transcripts (pri-miRNAs) and regulate numerous biological processes in eukaryotes. Due to the extreme heterogeneity of these structures, the initial processing sites of plant pri-miRNAs and the structural rules that determine their processing have been predicted for many miRNAs but remain elusive for others. Here we used semi-active DCL1 mutants and advanced degradome-sequencing strategies to accurately identify the initial processing sites for 147 of 326 previously annotated Arabidopsis miRNAs and to illustrate their associated pri-miRNA cleavage patterns. Elucidating the in vivo RNA secondary structures of 73 pri-miRNAs revealed that about 95% of them differ from in silico predictions, and that the revised structures offer clearer interpretation of the processing sites and patterns. Finally, DCL1 partners Serrate and HYL1 could synergistically and independently impact processing patterns and in vivo RNA secondary structures of pri-miRNAs. Together, our work sheds light on the precise processing mechanisms of plant pri-miRNAs.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , MicroARNs , ARN de Planta , Arabidopsis/genética , Arabidopsis/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ARN , Procesamiento Postranscripcional del ARN , Regulación de la Expresión Génica de las PlantasRESUMEN
BACKGROUND: RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3' untranslated regions (3' UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3' UTRs in gene expression in different organisms and/or contexts. RESULTS: Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3' UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3' UTR and, resultantly, a poorly structured 3' UTR. RNA decay assays indicate that poorly structured 3' UTRs could promote mRNA stability, whereas highly structured 3' UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3' UTRs' RSS and transcript accumulation in the transcriptomes of Arabidopsis, rice, and even human. Mechanistically, transcripts with highly structured 3' UTRs are preferentially degraded by 3'-5' exoribonuclease SOV and 5'-3' exoribonuclease XRN4, leading to decreased expression in Arabidopsis. Finally, we engineer different structured 3' UTRs to an endogenous FT gene and alter the FT-regulated flowering time in Arabidopsis. CONCLUSIONS: We conclude that highly structured 3' UTRs typically cause reduced accumulation of the harbored transcripts in Arabidopsis. This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3' UTRs' RSS to modify plant traits in agricultural production and mRNA stability in biotechnology.
Asunto(s)
Arabidopsis , Exorribonucleasas , Animales , Humanos , Regiones no Traducidas 3' , ARN Mensajero/genética , ARN Mensajero/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica , Mamíferos/genéticaRESUMEN
Intrinsically disordered proteins (IDPs) SAID1/2 are hypothetic dentin sialophosphoprotein-like proteins, but their true functions are unknown. Here, we identified SAID1/2 as negative regulators of SERRATE (SE), a core factor in miRNA biogenesis complex (microprocessor). Loss-of-function double mutants of said1; said2 caused pleiotropic developmental defects and thousands of differentially expressed genes that partially overlapped with those in se. said1; said2 also displayed increased assembly of microprocessor and elevated accumulation of microRNAs (miRNAs). Mechanistically, SAID1/2 promote pre-mRNA processing 4 kinase A-mediated phosphorylation of SE, causing its degradation in vivo. Unexpectedly, SAID1/2 have strong binding affinity to hairpin-structured pri-miRNAs and can sequester them from SE. Moreover, SAID1/2 directly inhibit pri-miRNA processing by microprocessor in vitro. Whereas SAID1/2 did not impact SE subcellular compartmentation, the proteins themselves exhibited liquid-liquid phase condensation that is nucleated on SE. Thus, we propose that SAID1/2 reduce miRNA production through hijacking pri-miRNAs to prevent microprocessor activity while promoting SE phosphorylation and its destabilization in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrínsecamente Desordenadas , MicroARNs , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ARN/metabolismo , Procesamiento Postranscripcional del ARN , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Photorespiratory hydrogen peroxide (H2 O2 ) plays key roles in pathogenesis responses by triggering the salicylic acid (SA) pathway in Arabidopsis. However, factors linking intracellular H2 O2 to activation of the SA pathway remain elusive. In this work, the catalase-deficient Arabidopsis mutant, cat2, was exploited to elucidate the impact of S-nitrosoglutathione reductase 1 (GSNOR1) on H2 O2 -dependent signalling pathways. Introducing the gsnor1-3 mutation into the cat2 background increased S-nitrosothiol levels and abolished cat2-triggered cell death, SA accumulation, and associated gene expression but had little additional effect on the major components of the ascorbate-glutathione system or glycolate oxidase activities. Differential transcriptome profiles between gsnor1-3 and cat2 gsnor1-3 together with damped ROS-triggered gene expression in cat2 gsnor1-3 further indicated that GSNOR1 acts to mediate the SA pathway downstream of H2 O2 . Up-regulation of GSNOR activity was compromised in cat2 cad2 and cat2 pad2 mutants in which glutathione accumulation was genetically prevented. Experiments with purified recombinant GSNOR revealed that the enzyme is posttranslationally regulated by direct denitrosation in a glutathione-dependent manner. Together, our findings identify GSNOR1-controlled nitrosation as a key factor in activation of the SA pathway by H2 O2 and reveal that glutathione is required to maintain this biological function.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Transducción de Señal , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Microscopía Confocal , Nitrosación , Estrés Oxidativo , Reguladores del Crecimiento de las Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácido Salicílico/metabolismoRESUMEN
There is growing evidence that hydrogen sulfide (H2S) is involved in many physiological processes in plants, but the role of H2S in dark-induced leaf senescence remains unknown. In this work, we found that H2S not only inhibited chlorophyll degradation but also caused the accumulation of photoreactive pheide a in detached leaves under extended darkness. Despite this, transcript levels of senescence-associated genes (SAGs) were less affected in H2S-treated detached leaves compared with those in H2S-untreated detached leaves. Furthermore, cell death/rapid bleaching occurred in both H2S-treated detached and attached leaves after transfer from extended darkness to light. Unlike the lack of effect of H2S on SAG transcripts in darkened detached leaves, exogenous H2S induced higher SAG transcript levels in attached leaves than untreated attached leaves. Genetic evidence further underlined the positive correlation between SAG expression in attached leaves and H2S. In addition, effects of H2S on SAG expression in attached leaves were compromised in the S-nitrosoglutathione reductase-deficient mutant, gsnor1. Taken together, our results suggest that H2S suppresses chlorophyll degradation of detached leaves by regulating a dark-dependent reaction, and that this gas positively modulates SAG expression in attached leaves under prolonged darkness in a GSNOR1-dependent manner.