key: cord-013243-1hj5clsw
authors: Brewer, Gary; Li, Mei-Ling; Tolbert, Blanton S.
title: Editorial for “Methods to characterize virus small RNAs and RNA structures”
date: 2020-10-16
journal: Methods
DOI: 10.1016/j.ymeth.2020.10.007
sha: 
doc_id: 13243
cord_uid: 1hj5clsw

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translation. In addition to vsRNA1, inhibition requires IRES-associated trans-acting factors (ITAFs), i.e., host proteins. Earlier work showed that stem-loop II (SL-II) in the EV-A71 IRES is a hot spot for association with host RNA-binding proteins, including hnRNP A1, mRNA decay factor AUF1, mRNA stability factor HuR, and RISC subunit Ago2 [4] . Li and Brewer [3; Li paper] describe methods for functional analyses of vsRNA1-mediated repression of IRES activity.

These include construction of bicistronic IRES-luciferase reporters, protein-RNA interaction assays, knockdown of RNA-binding proteins using RNA interference, and assessing effects of vsRNAs and host RNA-binding proteins on viral protein synthesis and replication. Importantly, these methods should be applicable to other virus-derived, small RNAs as well. The third article by Jiang et al. [5; Julkunen paper] in this section describes an in vitro transcription system to produce virus-specific ssRNAs and dsRNAs. ssRNAs are produced by traditional DNA-templated transcription using T7 DNA-dependent RNA polymerase. Of course, these templates can encode RNA corresponding to any viral genomic or subgenomic RNA squences. For synthesis of dsRNA, they employ the RNA-dependent RNA polymerase phi6 and a ssRNA template to produce dsRNA.

One of the advantages of this enzymatic approach is that it minimizes problems that can arise upon annealing two complementary RNA strands, e.g., secondary structure within a ssRNA due to selfannealing; and low yields of long dsRNAs. These RNAs can be subsequently modified (e.g., Here, the small molecule dimethyl sulfate (DMS) preferentially methylates unpaired or dynamic adenosine and cytosine residues within a viral RNA genome. Those modified residues are encoded as mutations during reverse transcription by the thermostable group II intron reverse transcriptase (TGIR-II) and subsequently detected by deep sequencing. The modification indices can be incorporated into RNA folding algorithms to determine experimental structures of viral genomes.

By comparison, the method described by Lukasiewicz and Contreras [12; Contreras paper] directly probes the accessibility of unpaired regions within RNA structure by using an antisense platform that has been engineered to express GFP only when hybridized to the appropriate RNA target. 

Methods for detection and study of virus derived small RNAs produced from the intramolecular base-pairing region of the picornavirus genome

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

Functional analyses of mammalian virus 5'UTR-derived, small RNAs that regulate virus translation

HuR and Ago2 bind the internal ribosomal entry site of enterovirus 71 and promote virus replication

In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses

From current knowledge to best practice: A primer on Viral diagnostics using deep sequencing of virus-derived small interfering RNAs (vsiRNAs) in infected plants

Potential functional pathways of plant RNA virus-derived small RNAs in a vector insect

Molecular characterization and RSV Co-infection of Nicotiana benthamiana with three distinct begomoviruses

Integrated approaches to reveal Mechanisms by which RNA viruses reprogram the cellular environment

Mapping the RNA structural landscape of viral genomes

Viral RNA structure analysis using DMS-MaPseq

Antisense probing of dynamic RNA structures

CLIP for studying protein-RNA interactions that regulate virus replication