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Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation., Competing Interests: Declaration of interests OSR is a member of the Molecular Cell Scientific Advisory Board and the Cell Reports Scientific Advisory Board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLS Tyr ) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLS Tyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLS Tyr and the 3' domain from a hordeivirus TLS Tyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLS Tyr s performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLS Tyr Finally, these structurally and phylogenetically divergent types of TLS Tyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures., (© 2024 Sherlock et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Non-structural protein 1 (Nsp1), which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from β-Coronaviruses (β-CoV) inhibits translation through ribosome binding. The C-terminal domain (CTD) of all β-CoV Nsp1s confers high-affinity ribosome binding despite low sequence conservation. Modeling of interactions of four Nsp1s with the ribosome identified the few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the β-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1's N-terminal "effector" domain. Finally, we show that a cis-acting viral RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aid future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2 and related human-pathogenic β-CoVs. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

All flaviviruses contain conserved RNA structures in the 3' untranslated region (3' UTR) that are important for flavivirus RNA replication, translation, and pathogenesis. Flaviviruses like Zika virus (ZIKV) contain multiple conserved RNA structures in the viral 3' UTR, including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus positive-strand genome replication, but the functional role of the flavivirus DB-1 structure and the mechanism by which it contributes to viral pathogenesis are not known. Using the recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones, termed ZIKV-TL.PK and ZIKV-p.2.5', which disrupt DB-1 tertiary folding. We found that viral positive-strand genome replication of both ZIKV DB-1 mutant clones is similar to wild-type (WT) ZIKV, but ZIKV DB-1 mutants exhibit significantly decreased cytopathic effect due to reduced caspase-3 activation. We next show that ZIKV DB-1 mutants exhibit decreased levels of sfRNA species compared to ZIKV-WT during infection. However, ZIKV DB-1 mutant 3' UTRs exhibit unchanged sfRNA biogenesis following XRN1 degradation in vitro . We also found that ZIKV DB-1 mutant virus (ZIKV-p.2.5') exhibited enhanced sensitivity to type I interferon treatment, and both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality due to tissue-specific attenuated viral replication in brain tissue of interferon type I/II receptor knockout mice. We propose that the flavivirus DB-1 RNA structure maintains sfRNA levels during infection despite maintained sfRNA biogenesis, and these results indicate that ZIKV DB-dependent maintenance of sfRNA levels support caspase-3-dependent, cytopathic effect, type I interferon resistance, and viral pathogenesis in mammalian cells and in a ZIKV murine model of disease. IMPORTANCE The group of viruses termed flaviviruses cause important disease throughout the world and include dengue virus, Zika virus, Japanese encephalitis virus, and many more. All of these flaviviruses have highly conserved RNA structures in the untranslated regions of the virus genome. One of the shared RNA structures, termed the dumbbell region, is not well studied, but mutations in this region are important for vaccine development. In this study, we made structure-informed targeted mutations in the Zika virus dumbbell region and studied the effect on the virus. We found that Zika virus dumbbell mutants are significantly weakened or attenuated due to a decreased ability to produce non-coding RNA that is needed to support infection, support virus-induced cell death, and support escape from the host immune system. These data show that targeted mutations in the flavivirus dumbbell RNA structure may be an important approach to develop future vaccine candidates., Competing Interests: The authors declare no conflict of interest.

The gene expression pathway from DNA sequence to functional protein is not as straightforward as simple depictions of the central dogma might suggest. Each step is highly regulated, with complex and only partially understood molecular mechanisms at play. Translation is one step where the "one gene-one protein" paradigm breaks down, as often a single mature eukaryotic mRNA leads to more than one protein product. One way this occurs is through translation reinitiation, in which a ribosome starts making protein from one initiation site, translates until it terminates at a stop codon, but then escapes normal recycling steps and subsequently reinitiates at a different downstream site. This process is now recognized as both important and widespread, but we are only beginning to understand the interplay of factors involved in termination, recycling, and initiation that cause reinitiation events. There appear to be several ways to subvert recycling to achieve productive reinitiation, different types of stresses or signals that trigger this process, and the mechanism may depend in part on where the event occurs in the body of an mRNA. This perspective reviews the unique characteristics and mechanisms of reinitiation events, highlights the similarities and differences between three major scenarios of reinitiation, and raises outstanding questions that are promising avenues for future research., (© 2023 Sherlock et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

While DNA and RNA helices often adopt the canonical B- or A-conformation, the fluid conformational landscape of nucleic acids allows for many higher energy states to be sampled. One such state is the Z-conformation of nucleic acids, which is unique in that it is left-handed and has a "zigzag" backbone. The Z-conformation is recognized and stabilized by Z-DNA/RNA binding domains called Zα domains. We recently demonstrated that a wide range of RNAs can adopt partial Z-conformations termed "A-Z junctions" upon binding to Zα and that the formation of such conformations may be dependent upon both sequence and context. In this chapter, we present general protocols for characterizing the binding of Zα domains to A-Z junction-forming RNAs for the purpose of determining the affinity and stoichiometry of interactions as well as the extent and location of Z-RNA formation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The p53 transcription factor is a master regulator of cellular stress responses inhibited by repressors such as MDM2 and the phosphatase PPM1D. Activation of p53 with pharmacological inhibitors of its repressors is being tested in clinical trials for cancer therapy, but efficacy has been limited by poor induction of tumor cell death. We demonstrate that dual inhibition of MDM2 and PPM1D induces apoptosis in multiple cancer cell types via amplification of the p53 transcriptional program through the eIF2α-ATF4 pathway. PPM1D inhibition induces phosphorylation of eIF2α, ATF4 accumulation, and ATF4-dependent enhancement of p53-dependent transactivation upon MDM2 inhibition. Dual inhibition of p53 repressors depletes heme and induces HRI-dependent eIF2α phosphorylation. Pharmacological induction of eIF2α phosphorylation synergizes with MDM2 inhibition to induce cell death and halt tumor growth in mice. These results demonstrate that PPM1D inhibits both the p53 network and the integrated stress response controlled by eIF2α-ATF4, with clear therapeutic implications., (© 2022. The Author(s).)

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients., Competing Interests: HP, MG, VZ, AG, CJ, JK, JD No competing interests declared, (© 2022, Park et al.)

Conformational dynamics are essential to macromolecular function. This is certainly true of RNA, whose ability to undergo programmed conformational dynamics is essential to create and regulate complex biological processes. However, methods to easily and simultaneously interrogate both the structure and conformational dynamics of fully functional RNAs in isolation and in complex with proteins have not historically been available. Due to its ability to image and classify single particles, cryogenic electron microscopy (cryo-EM) has the potential to address this gap and may be particularly amenable to exploring structural dynamics within the three-dimensional folds of biologically active RNAs. We discuss the possibilities and current limitations of applying cryo-EM to simultaneously study RNA structure and conformational dynamics, and present one example that illustrates this (as of yet) not fully realized potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of these misfolded states, but they are difficult to characterize because of their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophila group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.

Recent events have pushed RNA research into the spotlight. Continued discoveries of RNA with unexpected diverse functions in healthy and diseased cells, such as the role of RNA as both the source and countermeasure to a severe acute respiratory syndrome coronavirus 2 infection, are igniting a new passion for understanding this functionally and structurally versatile molecule. Although RNA structure is key to function, many foundational characteristics of RNA structure are misunderstood, and the default state of RNA is often thought of and depicted as a single floppy strand. The purpose of this perspective is to help adjust mental models, equipping the community to better use the fundamental aspects of RNA structural information in new mechanistic models, enhance experimental design to test these models, and refine data interpretation. We discuss six core observations focused on the inherent nature of RNA structure and how to incorporate these characteristics to better understand RNA structure. We also offer some ideas for future efforts to make validated RNA structural information available and readily used by all researchers.

Viruses require multifunctional structured RNAs to hijack their host’s biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo–electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)–like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.

What viral RNA genomes lack in size, they make up for in intricacy. Elaborate RNA structures embedded in viral genomes can hijack essential cellular mechanisms aiding virus propagation. Exoribonuclease-resistant RNAs (xrRNAs) are an emerging class of viral elements, which resist degradation by host cellular exoribonucleases to produce viral RNAs with diverse roles during infection. Detailed three-dimensional structural studies of xrRNAs from flaviviruses and a subset of plant viruses led to a mechanistic model in which xrRNAs block enzymatic digestion using a ring-like structure that encircles the 5' end of the resistant structure. In this mini-review, we describe the state of our understanding of the phylogenetic distribution of xrRNAs, their structures, and their conformational dynamics. Because xrRNAs have now been found in several major superfamilies of RNA viruses, they may represent a more widely used strategy than currently appreciated. Could xrRNAs represent a 'molecular clock' that would help us understand virus evolution and pathogenicity? The more we study xrRNAs in viruses, the closer we get to finding xrRNAs within cellular RNAs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some viral RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLS His ) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate processes important for infection, to include genome replication. As only five TLS His examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLS His , yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLS His RNAs. We confirmed new examples as authentic TLS His by demonstrating their ability to be histidylated in vitro, then used mutational analyses to imply a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structure and function in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs., (© 2021 Langeberg et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLS Val ) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLS Val have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLS Val We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLS Val substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection., (© 2021 Sherlock et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg 2+ ions, and a C + -G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond., (© 2021 Jones et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences., (© 2020 Steckelberg et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Viruses have developed innovative strategies to exploit the cellular machinery and overcome the antiviral defenses of the host, often using specifically structured RNA elements. Examples are found in the Flavivirus genus (in the family Flaviviridae ), where during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5' to 3' exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease-resistant RNA structure (xrRNA) located in the viral genome's 3' untranslated region (UTR). Although known to exist in several Flaviviridae genera, the full distribution and diversity of xrRNAs in this family were unknown. Using the recently solved high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNAs in the remaining three genera of Flaviviridae : Pegivirus , Pestivirus , and Hepacivirus We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Overall, we have identified the presence of xrRNA in all four genera of Flaviviridae , but not in all species. Our findings thus require adjustments of previous xrRNA classification schemes and expand the previously known distribution of xrRNA in Flaviviridae. IMPORTANCE The members of the Flaviviridae comprise one of the largest families of positive-sense single-stranded RNA (+ssRNA) and are divided into the Flavivirus , Pestivirus , Pegivirus , and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika virus, dengue virus, and Powassan virus. In these, a part of the RNA of the virus twists up into a distinct three-dimensional shape called an exoribonuclease-resistant RNA (xrRNA) that blocks the ability of the cell to "chew up" the viral RNA. Hence, part of the RNA of the virus remains intact, and this protected part is important for viral infection. These xrRNAs were known to occur in flaviviruses, but whether they existed in the other members of the family was not known. In this study, we identified a new subclass of xrRNA found not only in flaviviruses but also in the remaining three genera. The fact that these structured viral RNAs exist throughout the Flaviviridae family suggests they are important parts of the infection strategy of diverse pathogens, which could lead to new avenues of research., (Copyright © 2020 Szucs et al.)

Programmed -1 ribosomal frameshifts (-1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent -1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis -acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3' boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3' boundary would be defined by the pseudoknot's 3' end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3' boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol -1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol -1 PRF and define the frameshift site's 3' boundary., (© 2020 Thulson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.

Interleukin-37 (IL-37), a member of the IL-1 family of cytokines, is a fundamental suppressor of innate and acquired immunities. Here, we used an integrative approach that combines biophysical, biochemical, and biological studies to elucidate the unique characteristics of IL-37. Our studies reveal that single amino acid mutations at the IL-37 dimer interface that result in the stable formation of IL-37 monomers also remain monomeric at high micromolar concentrations and that these monomeric IL-37 forms comprise higher antiinflammatory activities than native IL-37 on multiple cell types. We find that, because native IL-37 forms dimers with nanomolar affinity, higher IL-37 only weakly suppresses downstream markers of inflammation whereas lower concentrations are more effective. We further show that IL-37 is a heparin binding protein that modulates this self-association and that the IL-37 dimers must block the activity of the IL-37 monomer. Specifically, native IL-37 at 2.5 nM reduces lipopolysaccharide (LPS)-induced vascular cell adhesion molecule (VCAM) protein levels by ∼50%, whereas the monomeric D73K mutant reduced VCAM by 90% at the same concentration. Compared with other members of the IL-1 family, both the N and the C termini of IL-37 are extended, and we show they are disordered in the context of the free protein. Furthermore, the presence of, at least, one of these extended termini is required for IL-37 suppressive activity. Based on these structural and biological studies, we present a model of IL-37 interactions that accounts for its mechanism in suppressing innate inflammation., Competing Interests: The authors declare no conflict of interest.

Viruses must co-opt the cellular translation machinery to produce progeny virions. Eukaryotic viruses have evolved a variety of ways to manipulate the cellular translation apparatus, in many cases using elegant RNA-centred strategies. Viral RNAs can alter or control every phase of protein synthesis and have diverse targets, mechanisms and structures. In addition, as cells attempt to limit infection by downregulating translation, some of these viral RNAs enable the virus to overcome this response or even take advantage of it to promote viral translation over cellular translation. In this Review, we present important examples of viral RNA-based strategies to exploit the cellular translation machinery. We describe what is understood of the structures and mechanisms of diverse viral RNA elements that alter or regulate translation, the advantages that are conferred to the virus and some of the major unknowns that provide motivation for further exploration.

Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus Dianthovirus Also, xrRNAs had been found only in the 3' untranslated regions (3'UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5' ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known. IMPORTANCE During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as "riboregulators" that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and "riboregulatory" RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions., (Copyright © 2018 Steckelberg et al.)

Communication between the 5' and 3' ends of mature eukaryotic mRNAs lies at the heart of gene regulation, likely arising at the same time as the eukaryotic lineage itself. Our view of how and why it occurs has been shaped by elegant experiments that led to nearly universal acceptance of the "closed-loop model." However, new observations suggest that this classic model needs to be reexamined, revised, and expanded. Here, we address fundamental questions about the closed-loop model and discuss how a growing understanding of mRNA structure, dynamics, and intermolecular interactions presents new experimental opportunities. We anticipate that the application of emerging methods will lead to expanded models that include the role of intrinsic mRNA structure and quantitative dynamic descriptions of 5'-3' proximity linked to the functional status of an mRNA and will better reflect the messy realities of the crowded and rapidly changing cellular environment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Structured RNA elements, programmed RNA conformational changes, and interactions between different RNA domains underlie many modes of regulating gene expression, mandating studies to understand the foundational principles that govern these phenomena. Exploring the structured 3' untranslated region (UTR) of a viral RNA, we discovered that different contexts of the 3'-UTR confer different abilities to enhance translation of an associated open reading frame. In one context, ribosome-induced conformational changes in a 'sensor' RNA domain affect a separate RNA 'functional' domain, altering translation efficiency. The structure of the entire 3'-UTR reveals that structurally distinct domains use a spine of continuously stacked bases and a strut-like linker to create a conduit for communication within the higher-order architecture. Thus, this 3'-UTR RNA illustrates how RNA can use programmed conformational changes to sense the translation status of an upstream open reading frame, then create a tuned functional response by communicating that information to other RNA elements.

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs., Competing Interests: The authors declare no conflict of interest.

Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5'-3' progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome's 3'-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that 'brace' against an enzyme's surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies.

Internal ribosome entry sites (IRESs) are important RNA-based translation initiation signals, critical for infection by many pathogenic viruses. The hepatitis C virus (HCV) IRES is the prototype for the type 3 IRESs and is also invaluable for exploring principles of eukaryotic translation initiation, in general. Current mechanistic models for the type 3 IRESs are useful but they also present paradoxes, including how they can function both with and without eukaryotic initiation factor (eIF) 2. We discovered that eIF1A is necessary for efficient activity where it stabilizes tRNA binding and inspects the codon-anticodon interaction, especially important in the IRES' eIF2-independent mode. These data support a model in which the IRES binds preassembled translation preinitiation complexes and remodels them to generate eukaryotic initiation complexes with bacterial-like features. This model explains previous data, reconciles eIF2-dependent and -independent pathways, and illustrates how RNA structure-based control can respond to changing cellular conditions., Competing Interests: The authors declare that no competing interests exist.

The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure that is responsible for ZIKV sfRNA production forms a complex fold that is likely found in many pathogenic flaviviruses. Mutations that disrupt the structure affect exonuclease resistance in vitro and sfRNA formation during infection. The complete ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies features that may modulate function in diverse flaviviruses., (Copyright © 2016, American Association for the Advancement of Science.)

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Communication between the 5' and 3' ends of a eukaryotic messenger RNA (mRNA) or viral genomic RNA is a ubiquitous and important strategy used to regulate gene expression. Although the canonical interaction between initiation factor proteins at the 5' end of an mRNA and proteins bound to the polyadenylate tail at the 3' end is well known, in fact there are many other strategies used in diverse ways. These strategies can involve "non-canonical" proteins, RNA structures, and direct RNA-RNA base-pairing between distal elements to achieve 5'-to-3' communication. Likewise, the communication induced by these interactions influences a variety of processes linked to the use and fate of the RNA that contains them. Recent studies are revealing how dynamic these interactions are, possibly changing in response to cellular conditions or to link various phases of the mRNA's life, from translation to decay. Thus, 5'-to-3' communication is about more than just making a closed circle; the RNA elements and associated proteins are key players in controlling gene expression at the post-transcriptional level., Competing Interests: The authors declare that they have no competing interests. No competing interests were disclosed. No competing interests were disclosed.

The packaging RNA (pRNA) found in the phi29 family of bacteriophage is an essential component of a powerful molecular motor used to package the phage's DNA genome into the capsid. The pRNA forms homomultimers mediated by intermolecular "kissing-loop" interactions, thus it is an example of the unusual phenomenon of a self-associating RNA that can form symmetric higher-order multimers. Previous research showed the pRNAs from phi29 family phages have diverse self-association properties and the kissing-loop interaction is not the sole structural element dictating multimerization. We found that a 3-way junction (3wj) within each pRNA, despite not making direct intermolecular contacts, plays important roles in stabilizing the intermolecular interactions and dictating the size of the multimer formed (dimer, trimer, etc.). Specifically, the 3wj in the pRNA from phage M2 appears to favor a different conformation compared to the 3wj in the phi29 pRNA, and the M2 junction facilitates formation of a higher-order multimer that is more thermostable. This behavior provides insights into the fundamental principles of RNA self-association, and additionally may be useful to engineer fine-tuned properties into pRNAs for nanotechnology.

Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi's sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Solving a novel RNA structure by x-ray crystallography requires a means to obtain initial phase estimates. This is a challenge because many of the tools available for solving protein structures are not available for RNA. We have developed a reliable means to use hexammine cations to address this challenge. The process involves engineering the RNA to introduce a reliable hexammine binding site into the structure, then soaking crystals of these RNAs with an iridium (III) or cobalt (III) compound in a "directed soaking" strategy. Diffraction data obtained from these crystals then can be used in SAD or MAD phasing. In many cases, suitable derivatives can be obtained by soaking the hexammine into RNA crystals that have not been engineered. Considerations for using this method and example protocols are presented.

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs.

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3'UTR that interact with Xrn1 to halt processive 5' to 3' decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3' UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3' UTR architecture, sfRNA production, and host adaptation.

The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage's DNA genome. The pRNA forms higher-order multimers by intermolecular "kissing" interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA's junction, we propose a model for how different junctions affect self-association., (© 2014 Hao and Kieft; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles, and this can be conferred by a complex three-dimensional structure. Such multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. To address this gap it is useful to study examples from single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3' end of the turnip yellow mosaic virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic (g)RNA, but also interacts with other structures in the 3' untranslated region of the gRNA, contains the promoter for negative-strand synthesis, and influences several infection-critical processes. TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is 'two faced': one face closely mimics tRNA and drives aminoacylation, the other face diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes.