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1. RNA regulatory processes in RNA virus biology

   https://doi.org/10.1002/wrna.1536  

   Cross, Shaun T. ; Michalski, Daniel ; Miller, Megan R. ; Wilusz, Jeffrey ( April 2019 , WIREs RNA)

   Numerous post‐transcriptional RNA processes play a major role in regulating the quantity, quality and diversity of gene expression in the cell. These include RNA processing events such as capping, splicing, polyadenylation and modification, but also aspects such as RNA localization, decay, translation, and non‐coding RNA‐associated regulation. The interface between the transcripts of RNA viruses and the various RNA regulatory processes in the cell, therefore, has high potential to significantly impact virus gene expression, regulation, cytopathology and pathogenesis. Furthermore, understanding RNA biology from the perspective of an RNA virus can shed considerable light on the broad impact of these post‐transcriptional processes in cell biology. Thus the goal of this article is to provide an overview of the richness of cellular RNA biology and how RNA viruses use, usurp and/or avoid the associated machinery to impact the outcome of infection.

   This article is categorized under:

   RNA in Disease and Development > RNA in Disease

    

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2. Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

   https://doi.org/10.1002/cpnc.113  

   Wu, Ying ; Zheng, Ya Ying ; Lin, Qishan ; Sheng, Jia ( August 2020 , Current Protocols in Nucleic Acid Chemistry)

   This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides

   Basic Protocol 2: Digestion of RNA samples extracted from cells

   Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry

    

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3. Translational gene regulation in plants: A green new deal

   https://doi.org/10.1002/wrna.1597  

   Urquidi Camacho, Ricardo A. ; Lokdarshi, Ansul ; von Arnim, Albrecht G. ( May 2020 , WIREs RNA)

   The molecular machinery for protein synthesis is profoundly similar between plants and other eukaryotes. Mechanisms of translational gene regulation are embedded into the broader network of RNA‐level processes including RNA quality control and RNA turnover. However, over eons of their separate history, plants acquired new components, dropped others, and generally evolved an alternate way of making the parts list of protein synthesis work. Research over the past 5 years has unveiled how plants utilize translational control to defend themselves against viruses, regulate translation in response to metabolites, and reversibly adjust translation to a wide variety of environmental parameters. Moreover, during seed and pollen development plants make use of RNA granules and other translational controls to underpin developmental transitions between quiescent and metabolically active stages. The economics of resource allocation over the daily light–dark cycle also include controls over cellular protein synthesis. Important new insights into translational control on cytosolic ribosomes continue to emerge from studies of translational control mechanisms in viruses. Finally, sketches of coherent signaling pathways that connect external stimuli with a translational response are emerging, anchored in part around TOR and GCN2 kinase signaling networks. These again reveal some mechanisms that are familiar and others that are different from other eukaryotes, motivating deeper studies on translational control in plants.

   This article is categorized under:

   Translation > Translation Regulation

   RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

   RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications

    

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4. Preparation of Mammalian Nascent RNA for Long Read Sequencing

   https://doi.org/10.1002/cpmb.128  

   Reimer, Kirsten A. ; Neugebauer, Karla M. ( October 2020 , Current Protocols in Molecular Biology)

   Long read sequencing technologies now allow high‐quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single‐nucleotide‐resolution information about co‐transcriptional RNA processing events—e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin‐associated RNA, as well as how to deplete poly(A)⁺RNA and rRNA, and, finally, how to generate a full‐length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi‐intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Subcellular fractionation

   Basic Protocol 2: Nascent RNA isolation and adapter ligation

   Basic Protocol 3: cDNA amplicon preparation

    

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5. Unwinding the roles of RNA helicase MOV10

   https://doi.org/10.1002/wrna.1682  

   Nawaz, Aatiqa ; Shilikbay, Temirlan ; Skariah, Geena ; Ceman, Stephanie ( July 2021 , WIREs RNA)

   MOV10 is an RNA helicase that associates with the RNA‐induced silencing complex component Argonaute (AGO), likely resolving RNA secondary structures. MOV10 also binds the Fragile X mental retardation protein to block AGO2 binding at some sites and associates with UPF1, a principal component of the nonsense‐mediated RNA decay pathway. MOV10 is widely expressed and has a key role in the cellular response to viral infection and in suppressing retrotransposition. Posttranslational modifications of MOV10 include ubiquitination, which leads to stimulation‐dependent degradation, and phosphorylation, which has an unknown function. MOV10 localizes to the nucleus and/or cytoplasm in a cell type‐specific and developmental stage‐specific manner. Knockout ofMov10leads to embryonic lethality, underscoring an important role in development where it is required for the completion of gastrulation. MOV10 is expressed throughout the organism; however, most studies have focused on germline cells and neurons. In the testes, the knockdown ofMov10disrupts proliferation of spermatogonial progenitor cells. In brain, MOV10 is significantly elevated postnatally and binds mRNAs encoding cytoskeleton and neuron projection proteins, suggesting an important role in neuronal architecture. HeterozygousMov10mutant mice are hyperactive and anxious and their cultured hippocampal neurons have reduced dendritic arborization. Zygotic knockdown ofMov10inXenopus laeviscauses abnormal head and eye development and mislocalization of neuronal precursors in the brain. Thus, MOV10 plays a vital role during development, defense against viral infection and in neuronal development and function: its many roles and regulation are only beginning to be unraveled.

   This article is categorized under:

   RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

   RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications

    

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