RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

Marmisollé, Facundo E.; García, María Laura; Reyes, Carina A.

doi:10.1186/s13007-018-0276-9

Cited by 18 publications

(21 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A modified RIP-seq assay was developed for the detection of RNAs of heterologous origin in plants and applied to transiently expressed nuclear epitope-containing proteins in Nicotiana benthamiana , but to-date this method has not been used for its intended purpose of detecting viral RNAs in plant cells [ 49 ].…”

Section: Overview Of Methods To Detect Rna-protein Interactions Anmentioning

confidence: 99%

“…Viral genomes rely on many RBPs of host origin for their replication and dissemination [ 49 ]. Consequently, host RBPs play a role in defense against viral pathogens; the Arabidopsis pumilio RNA binding protein 5 (APUM5) provides protection against cucumber mosaic virus (CMV) by directly binding to the 3’ UTR of CMV RNAs to repress CMV replication [ 4 , 132 ].…”

Section: Rna-protein Interactions In Plants As Unearthed By Classimentioning

confidence: 99%

See 1 more Smart Citation

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

Burjoski

Reddy

2021

IJMS

View full text Add to dashboard Cite

RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA–protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA–protein interactions in plants thus far and highlight the understanding of plant RNA–protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA–protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA–protein interactions for the study of gene regulation and RNA biology in plants.

show abstract

Section: Overview Of Methods To Detect Rna-protein Interactions Anmentioning

confidence: 99%

Section: Rna-protein Interactions In Plants As Unearthed By Classimentioning

confidence: 99%

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

Burjoski

Reddy

2021

IJMS

View full text Add to dashboard Cite

show abstract

“…The fold change of each RIP reaction from RT-qPCR data was calculated via 2-△△CT method as previously with minor modification (42) and the formulas see below. All the Ct value of each specimen (FLAG-tagged NS3, FLAG-tagged NS2B or FLAG non-fused blank pcDNA3.1) was normalized with the Input RNA to eliminate the possible differences in RNA samples preparation (△Ct).…”

Section: Methodsmentioning

confidence: 99%

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Jiang

Zhao

Bai

et al. 2019

Preprint

View full text Add to dashboard Cite

27Previous studies revealed that Japanese encephalitis virus (JEV) infection alters the 28 expression of miRNA in central nervous system (CNS). However, the mechanism of JEV 29 infection contributes to the regulation of miRNAs in CNS remain obscure. Here, we found 30 that a global degradation of mature miRNA in mouse brain and neuroblastoma cells after JEV 31 infection. In additional, the integrative analysis of miRNAs and mRNAs suggests that those 32 down-regulated miRNAs are primarily targeted inflammation genes and the miR-466d-3p 33 target the IL-1β which in the middle of those inflammation genes. Transfection of 34 miR-466d-3p decreased the IL-1β expression and inhibited the JEV replication in NA cells. 35 Interestingly, the miR-466d-3p level increased after JEV infection in the presence of 36 cycloheximide, which indicated that viral protein expression reduces miR-466d-3p. 37Therefore, we generated all the JEV coding protein and demonstrated that NS3 is a potent 38 miRNA suppressor. Furthermore, the NS3 of ZIKA virus, WNV, DENV1 and DENV2 also 39 decreased the expression of miR-466d-3p. The in vitro unwinding assay demonstrated that the 40 NS3 could unwind the pre-miR-466d and induce the disfunction of miRNA. Using 41 computational models and RNA immunoprecipitation assay, we found that arginine-rich 42 domains of NS3 are critical for pre-miRNA binding and the degradation of host miRNAs. 43Importantly, site-directed mutagenesis of conserved residues revealed that R226G and 44 R202W significantly reduced the binding affinity and degradation of pre-miR-466d. 45 Together, these results extend the helicase of Flavivirus function beyond unwinding duplex 46 RNA to the decay of pre-miRNAs, which provides a new mechanism of NS3 in regulating 47 miRNA pathways and promoting the neuroinflammation. 48 49 Author Summary 50 Host miRNAs had been reported to regulate JEV induced inflammation in central nervous 51 system. We found that the NS3 of JEV can reduce most of host miRNA expression. The 52 helicase region of the NS3 specifically binds to precursors of miRNA and lead to incorrect 53 unwinding of precursors of miRNAs which inhibits the function of miRNAs. This observation 54 leads to two major findings. First, we identified the miR-466d-3p targets to the host IL-1β and 55 E protein of JEV, and NS3 degrades the miR-466d-3p to promote the brain inflammation and 56 viral replication. Second, we proved that the arginine on the helicase of NS3 is the main 57 miRNA binding sites, and the miRNA degradation by NS3 was abolished when the R226 and 58 R202 were mutated on the NS3. These findings were also confirmed with NS3 of ZIKA virus, 59WNV and DENV which could decrease the expression level of miR-466d-3p to enhance the 60 inflammation. Our study provides new insights into the molecular mechanism of encephalitis 61 caused by JEV, and reveals several amino acid sites to further attenuate the JEV vaccine. 62 63 65 JEV is a single-stranded, positive-sense RNA virus belonging to flavivirus of the Flaviviridae 66 family...

show abstract

“…Este protocolo se puso a punto durante esta tesis y fue utilizado para el estudio de pre-miARNs unidos a proteínas mostrado en el Capítulo 2 (Marmisolle et al, 2018; trabajo adjunto al final de la tesis). Los pasos a seguir se detallan a continuación y se esquematizan en la Figura 53:…”

Section: II N Ensayos De Ripunclassified

“…Se optimizó la metodología de RIP utilizando extractos de hojas agroinfiltradas con las construcciones mencionadas (Figura 27) (detalles de la metodología en la sección V.II.N. de Materiales y Métodos; Anexo:Marmisolle et al, 2018).La expresión transitoria de las proteínas ensayadas se chequeó por microscopía bajo luz UV y alcanzó su máximo nivel a los 3 días post-agroinfiltración (dpa) (Figura 28. A-D).…”

unclassified

Estudio de los mecanismos de procesamiento de microARNs en plantas de naranjo dulce infectadas con el virus de la Psorosis de los cítricos

Marmisollé¹

View full text Add to dashboard Cite

En este trabajo de tesis, se utilizó el virus de la psorosis de los cítricos (CPsV), un virus ssRNA de sentido negativo, sin envoltura, tripartito que codifica 4 proteínas: una replicasa (RdRp, 280kDa), una proteína pequeña de 24kDa (24K), una aspartil proteasa (54kDa) y una proteína de cubierta (48 kDa). En los últimos años, se ha profundizado en el conocimiento de las funciones y localizaciones sub-celulares de las proteínas de CPsV por parte de nuestro grupo. Se ha podido caracterizar a dos de sus proteínas (54KCPsV y 24KCPsV) como supresoras virales, aunque los mecanismos por los cuales llevan a cabo su función han sido poco explorados. En este trabajo de Tesis nos propusimos investigar aspectos concernientes a la biogénesis y acumulación de miARNs de C. sinensis y su relación con la infección viral. De esta forma, nos focalizamos en el estudio del mecanismo de procesamiento de precursores de miARNs de naranjo dulce y su alteración mediada por proteínas del virus de la psorosis de los cítricos. El trabajo de dividió en 4 capítulos: En el capítulo 1 nos propusimos caracterizar a nivel de secuencia y estructura 10 familias de pre-miARNs de C. sinensis: se analizaron las familias de precursores de 10 miARNs de C. sinensis, en relación a su secuencia y estructura. Se compararon los determinantes estructurales encontrados con las respectivas familias de A. thaliana y se propusieron mecanismos de procesamiento para cada uno de ellos. El capítulo 2, tuvo como objetivo estudiar la acumulación de pre-miARNs en muestras provenientes de plantas de C. sinensis infectadas con CPsV y su interacción con proteínas virales. Se demostró que la acumulación de 6 pre-miARNs conservados en C. sinensis aumentan su acumulación en plantas infectadas con CPsV respecto de las muestras sin infectar y que estos cambios están relacionados con la severidad de los síntomas. Estos resultados, a su vez, se correlacionan con la reducción de las especies maduras reportadas previamente (Reyes et al., 2016) y con el aumento de los transcriptos targets de estos miARNs que serán analizados en el Capítulo 3. Se observó una clara alteración en la biogénesis de miARNs mediante ensayos de RIP, lo que podría suponer una interacción de las proteínas virales de CPsV con alguna de las proteínas accesorias del procesamiento de miARNs durante la infección, limitando su función y por consiguiente afectando el procesamiento de dichos miARNs. Para el capítulo 3, tuvimos como objetivo estudiar la acumulación de transcriptos targets de miARNs y su relación con la sintomatología en plantas de C. sinensis infectadas con CPsV. Con nuestro trabajo, pudimos predecir bioinformáticamente 10 genes targets correspondientes a cuatro miARNs de C. sinensis que no habían sido validados en reportes previos. A su vez, analizamos su acumulación durante la infección y pudimos asociar aspectos relacionados a la sintomatología causada por la enfermedad. Finalmente, el capítulo 4 se centró en analizar la interacción entre la proteína 24KCPsV y proteínas de la maquinaria de procesamiento de miARNs. Los resultados obtenidos indican que la proteína viral co-localiza con las 3 proteínas de maquinaria evaluadas (DCL1, HYL1 y SE) y, a su vez, se ha podido corroborar la interacción de 24KCPsV con las proteínas HYL1At y SEAt por las dos metodologías utilizadas, CoIP y BiFC. Estos resultados confirman la interferencia de 24KCPsV con la vía de biogénesis de miARNs explicada por la interacción proteína-proteína o proteína-ARN.

show abstract

RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

Cited by 18 publications

References 40 publications

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Estudio de los mecanismos de procesamiento de microARNs en plantas de naranjo dulce infectadas con el virus de la Psorosis de los cítricos

Contact Info

Product

Resources

About