The West Nile virus genome harbors essential riboregulatory elements with conserved and host-specific functional roles.

West Nile virus (WNV) is an arthropod-borne, positive-sense RNA virus that poses an increasing global threat due to warming climates and lack of effective therapeutics. Like other enzootic viruses, little is known about how host context affects the structure of the full-length RNA genome. Here, we report a complete secondary structure of the entire WNV genome within infected mammalian and arthropod cell lines. Our analysis affords structural insights into multiple, conserved aspects of flaviviral biology. We show that the WNV genome folds with minimal host dependence, and we prioritize well-folded regions for functional validation using structural homology between hosts as a guide. Using structure-disrupting, antisense locked nucleic acids, we then demonstrate that the WNV genome contains riboregulatory structures with conserved and host-specific functional roles. These results reveal promising RNA drug targets within flaviviral genomes, and they highlight the therapeutic potential of ASO-LNAs as both WNV-specific and pan-flaviviral therapeutic agents.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

Progress in 50 years of viroid research-Molecular structure, pathogenicity, and host adaptation.

Viroids are non-encapsidated, single-stranded, circular RNAs consisting of 246-434 nucleotides. Despite their non-protein-encoding RNA nature, viroids replicate autonomously in host cells. To date, more than 25 diseases in more than 15 crops, including vegetables, fruit trees, and flowers, have been reported. Some are pathogenic but others replicate without eliciting disease. Viroids were shown to have one of the fundamental attributes of life to adapt to environments according to Darwinian selection, and they are likely to be living fossils that have survived from the pre-cellular RNA world. In 50 years of research since their discovery, it was revealed that viroids invade host cells, replicate in nuclei or chloroplasts, and undergo nucleotide mutation in the process of adapting to new host environments. It was also demonstrated that structural motifs in viroid RNAs exert different levels of pathogenicity by interacting with various host factors. Despite their small size, the molecular mechanism of viroid pathogenicity turned out to be more complex than first thought.

Rapid enzymatic synthesis of long RNA polymers: A simple protocol to generate RNA libraries with random sequences.

RNA aptamers have several advantages over DNA aptamers due to their propensity to fold into three-dimensional structures. However, the synthesis of large RNA libraries remains a challenge as it requires more precautions to conserve their functional integrity, especially when such libraries are intended for aptamers or ribozymes selection. Here, we present an enzymatic method that enables the rapid synthesis of RNA polymers thanks to the efficient incorporation of ribonucleotides (NTPs) as well as chemically modified ribonucleotides by human DNA polymerase Theta (θ) mutants. These mutants have the ability to generate long single-stranded RNA polynucleotides of random sequences due to their improved template-free terminal nucleotidyltransferase activity. Here we describe the detailed protocols to produce large and diverse libraries of RNA, to make them ready to use in repeated cycles of Systematic Evolution of Ligands by Exponential enrichment (SELEX) and to synthesize C2'-modified nucleic acids with higher nuclease resistance.

The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle.

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

Effect of UV Radiation on Fluorescent RNA Aptamers' Functional and Templating Ability.

Damage from ultraviolet (UV) radiation was likely to be an important selection pressure during the origin of life. RNA is believed to have been central to the origin of life and might form the basis for simple synthetic cells. Although photodamage of DNA has been extensively studied, photodamage is highly dependent on local molecular context, and damage to functional RNAs has been relatively under-studied. We irradiated two fluorescent RNA aptamers and monitored the loss of activity, folding, and the kinetics of lesion accumulation. The loss of activity differed depending on the aptamer, with the Spinach2 aptamer retaining substantial activity after long exposure times. The binding pocket was particularly susceptible to damage, and melting of the duplex regions increased susceptibility; this is consistent with the view that duplex formation is protective. At the same time, susceptibility varied greatly depending on context, thus emphasizing the importance of studying many different RNAs to understand UV hardiness.

Ribosomal Peptides and Small Proteins on the Rise.

Genetically encoded and ribosomally synthesised peptides and small proteins act as important regulators in fundamental cellular processes, including gene expression, development, signalling and metabolism. Moreover, they also play a crucial role in eukaryotic and prokaryotic defence against microorganisms. Extremely diverse in size and structure, they are often subject to extensive post-translational modification. Recent technological advances are now allowing the analysis of the whole cellular transcriptome and proteome, revealing the presence of hundreds of long-overlooked alternative and short open reading frames (short ORFs, or sORFs) in mRNA and supposedly noncoding RNAs. However, in many instances the biological roles of their translational products remain to be elucidated. Here we provide an overview on the intriguing structural and functional diversity of ribosomally synthesised peptides and newly discovered peptides and small proteins.

cncRNAs: Bi-functional RNAs with protein coding and non-coding functions.

For many decades, the major function of mRNA was thought to be to provide protein-coding information embedded in the genome. The advent of high-throughput sequencing has led to the discovery of pervasive transcription of eukaryotic genomes and opened the world of RNA-mediated gene regulation. Many regulatory RNAs have been found to be incapable of protein coding and are hence termed as non-coding RNAs (ncRNAs). However, studies in recent years have shown that several previously annotated non-coding RNAs have the potential to encode proteins, and conversely, some coding RNAs have regulatory functions independent of the protein they encode. Such bi-functional RNAs, with both protein coding and non-coding functions, which we term as 'cncRNAs', have emerged as new players in cellular systems. Here, we describe the functions of some cncRNAs identified from bacteria to humans. Because the functions of many RNAs across genomes remains unclear, we propose that RNAs be classified as coding, non-coding or both only after careful analysis of their functions.

What is an RNA? A top layer for RNA classification.

Every ribonucleic acid begins its cellular life as a transcript. If the transcript or its processing product has a function it should be regarded an RNA. Nonfunctional transcripts, by-products from processing, degradation intermediates, even those originating from (functional) RNAs, and non-functional products of transcriptional gene regulation accomplished via the act of transcription, as well as stochastic (co)transcripts could simply be addressed as transcripts (class 0). The copious functional RNAs (class I), often maturing after one or more processing steps, already are systematized into ever expanding sub-classifications ranging from micro RNAs to rRNAs. Established sub-classifications addressing a wide functional diversity remain unaffected. mRNAs (class II) are distinct from any other RNA by virtue of their potential to be translated into (poly)peptide(s) on ribosomes. We are not proposing a novel RNA classification, but wish to add a basic concept with existing terminology (transcript, RNA, and mRNA) that should serve as an additional framework for carefully delineating RNA function from an avalanche of RNA sequencing data. At the same time, this top level hierarchical model should illuminate important principles of RNA evolution and biology thus heightening our awareness that in biology boundaries and categorizations are typically fuzzy.

RNA Aptamers as Molecular Tools to Study the Functionality of the Hepatitis C Virus CRE Region.

BACKGROUND: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. METHODS: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. RESULTS: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. CONCLUSIONS: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes.

Malignant transformation of colonic epithelial cells by a colon-derived long noncoding RNA.

Recent progress has been made in the identification of protein-coding genes and miRNAs that are expressed in and alter the behavior of colonic epithelia. However, the role of long non-coding RNAs (lncRNAs) in colonic homeostasis is just beginning to be explored. By gene expression profiling of post-mitotic, differentiated tops and proliferative, progenitor-compartment bottoms of microdissected adult mouse colonic crypts, we identified several lncRNAs more highly expressed in crypt bottoms. One identified lncRNA, designated non-coding Nras functional RNA (ncNRFR), resides within the Nras locus but appears to be independent of the Nras coding transcript. Stable overexpression of ncNRFR in non-transformed, conditionally immortalized mouse colonocytes results in malignant transformation, as determined by growth in soft agar and formation of highly invasive tumors in nude mice. Moreover, ncNRFR appears to inhibit the function of the tumor suppressor let-7. These results suggest precise regulation of ncNRFR is necessary for proper cell growth in the colonic crypt, and its misregulation results in neoplastic transformation.

Non-coding RNAs and functional RNA elements in Thermus thermophilus.

To complete the ThermusQ database, small non-coding RNAs (ncRNAs) and functional RNA elements found in Thermus thermophilus were summarized with annotations. The well-known three ncRNAs, M1 RNA, tmRNA and SRP RNA, were annotated as ttj8_nc001 to ttj8_nc003, and 10 novel RNAs were annotated as ttj8_nc004 to ttj8_nc013. Antisense RNAs for some ORFs were annotated as ttj8_EST00001 to ttj8_EST00006. In addition, a set of conserved sequences found in T. thermophilus HB27 were also described.

Functional nucleic acids as potent therapeutics against SARS-CoV-2 infection.

The COVID-19 pandemic has posed a severe threat to human life and the global economy. Although conventional treatments, including vaccines, antibodies, and small-molecule inhibitors, have been broadly developed, they usually fall behind the constant mutation of SARS-CoV-2, due to the long screening process and high production cost. Functional nucleic acid (FNA)-based therapeutics are a newly emerging promising means against COVID-19, considering their timely adaption to different mutants and easy design for broad-spectrum virus inhibition. In this review, we survey typical FNA-related therapeutics against SARS-CoV-2 infection, including aptamers, aptamer-integrated DNA frameworks, functional RNA, and CRISPR-Cas technology. We first introduce the pathogenesis, transmission, and evolution of SARS-CoV-2, then analyze the existing therapeutic and prophylactic strategies, including their pros and cons. Subsequently, the FNAs are recommended as potent alternative therapeutics from their screening process and controllable engineering to effective neutralization. Finally, we put forward the remaining challenges of the existing field and sketch out the future development directions.

Generation of Functional-RNA Arrays by In Vitro Transcription and In Situ RNA Capture for the Detection of RNA-RNA Interactions.

RNA performs a wide variety of vital cellular functions. These functions typically require interactions with other biological macromolecules, often as part of an intricate communication network. High-throughput techniques capable of analyzing RNA-based interactions are therefore essential. Functional-RNA arrays address this need, providing the capability of performing hundreds of miniature assays in parallel. Here we describe a method to generate functional-RNA arrays using in vitro transcription of a DNA template array and in situ RNA capture. We also suggest how functional-RNA arrays could be applied to investigating RNA-RNA interactions.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer.

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5'UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5'UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

A New Specific and Sensitive RT-qPCR Method Based on Splinted 5' Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs.

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs-the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5' Cytosine, C-doRNA-and streamline their analysis, we developed a new specific and sensitive splinted 5' ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5' end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.

Regulatory noncoding RNAs: functions and applications in health and disease.

Science is facing a new RNA world that is shaping our knowledge, and we are discovering a new horizon in molecular biology. New technologies revealed thousands and thousands of new RNAs, most of them located in what was once known as the "dark matter of DNA". They are functional regulatory RNAs and do not code for proteins, and they orchestrate the cellular function according to the changes needed. These noncoding RNAs are ubiquitous, and they are present from viruses to humans. In this Virtual Issue, The FEBS Journal features a collection of recent articles on long noncoding RNAs, microRNAs, and circular RNAs. It gives a broad perspective regarding their role in vascular diseases, ocular diseases, immune cell development and homeostasis, inflammation, production of extracellular matrix, and cancer. Furthermore, review-type articles highlight the potential use of noncoding RNAs in a wide range of applications.

Understanding small ORF diversity through a comprehensive transcription feature classification.

Small open reading frames (small ORFs/sORFs/smORFs) are potentially coding sequences smaller than 100 codons that have historically been considered junk DNA by gene prediction software and in annotation screening; however, the advent of next-generation sequencing has contributed to the deeper investigation of junk DNA regions and their transcription products, resulting in the emergence of smORFs as a new focus of interest in systems biology. Several smORF peptides were recently reported in non-canonical mRNAs as new players in numerous biological contexts; however, their relevance is still overlooked in coding potential analysis. Hence, this review proposes a smORF classification based on transcriptional features, discussing the most promising approaches to investigate smORFs based on their different characteristics. First, smORFs were divided into non-expressed (intergenic) and expressed (genic) smORFs. Second, genic smORFs were classified as smORFs located in non-coding RNAs (ncRNAs) or canonical mRNAs. Finally, smORFs in ncRNAs were further subdivided into sequences located in small or long RNAs, whereas smORFs located in canonical mRNAs were subdivided into several specific classes depending on their localization along the gene. We hope that this review provides new insights into large-scale annotations and reinforces the role of smORFs as essential components of a hidden coding DNA world.

Coding potential and sequence conservation of SARS-CoV-2 and related animal viruses.

In December 2019, a novel human-infecting coronavirus (SARS-CoV-2) was recognized in China. In a few months, SARS-CoV-2 has caused thousands of disease cases and deaths in several countries. Phylogenetic analyses indicated that SARS-CoV-2 clusters with SARS-CoV in the Sarbecovirus subgenus and viruses related to SARS-CoV-2 were identified from bats and pangolins. Coronaviruses have long and complex genomes with high plasticity in terms of gene content. To date, the coding potential of SARS-CoV-2 remains partially unknown. We thus used available sequences of bat and pangolin viruses to determine the selective events that shaped the genome structure of SARS-CoV-2 and to assess its coding potential. By searching for signals of significantly reduced variability at synonymous sites (dS), we identified six genomic regions, one of these corresponding to the programmed -1 ribosomal frameshift. The most prominent signal of dS reduction was observed within the E gene. A genome-wide analysis of conserved RNA structures indicated that this region harbors a putative functional RNA element that is shared with the SARS-CoV lineage. Additional signals of reduced dS indicated the presence of internal ORFs. Whereas the presence ORF9a (internal to N) was previously proposed by homology with a well characterized protein of SARS-CoV, ORF3h (for hypothetical, within ORF3a) was not previously described. The predicted product of ORF3h has 90% identity with the corresponding predicted product of SARS-CoV and displays features suggestive of a viroporin. Finally, analysis of the putative ORF10 revealed high dN/dS (3.82) in SARS-CoV-2 and related coronaviruses. In the SARS-CoV lineage, the ORF is predicted to encode a truncated protein and is neutrally evolving. These data suggest that ORF10 encodes a functional protein in SARS-CoV-2 and that positive selection is driving its evolution. Experimental analyses will be necessary to validate and characterize the coding and non-coding functional elements we identified.

Two Examples of RNA Aptamers with Antiviral Activity. Are Aptamers the Wished Antiviral Drugs?

The current Covid-19 pandemic has pointed out some major deficiencies of the even most advanced societies to fight against viral RNA infections. Once more, it has been demonstrated that there is a lack of efficient drugs to control RNA viruses. Aptamers are efficient ligands of a great variety of molecules including proteins and nucleic acids. Their specificity and mechanism of action make them very promising molecules for interfering with the function encoded in viral RNA genomes. RNA viruses store essential information in conserved structural genomic RNA elements that promote important steps for the consecution of the infective cycle. This work describes two well documented examples of RNA aptamers with antiviral activity against highly conserved structural domains of the HIV-1 and HCV RNA genome, respectively, performed in our laboratory. They are two good examples that illustrate the potential of the aptamers to fill the therapeutic gaps in the fight against RNA viruses.