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 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.
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.
Communication between the two ends of mRNA is key to its fate. But how does it occur? Is it mediated by the so-called “closed loop” or is the inherent structure of RNA sufficient for this process? Vicens et al. re-examine current views, highlight new evidence, and suggest opportunities for future research.
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-tRNAfMet) into the P site for start codon recognition.
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•Structures of the 30S ribosomal subunit with initiation factors, tRNA and mRNA•IF3 helps to position the correct start codon in the P site before binding of tRNA•Large-scale conformational changes of IF3 and tRNA are observed•IF3 movements facilitate the accommodation of initiator tRNA in P site
Bacterial ribosomes can start scanning for start codons only after undergoing large scale conformational changes governed by three key initiation factors.
In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide ‘cap’ on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic ...viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES–ribosome complexes are revealing the structural basis of viral IRES’ ‘hijacking’ of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes.
Flaviviruses are emerging human pathogens and worldwide health threats. During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5′→3′ host cell ...exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly conserved nucleotides. The RNA's three-dimensional topology creates a ringlike conformation, with the 5′ end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.
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•RNA structure comprises conformational ensembles that are difficult to explore directly using traditional methods.•Cryo-electron microscopy (cryo-EM) can, in theory, be used to ...simultaneously explore RNA 3D structure and its conformational dynamics, but there are challenges.•A tRNA-mimicking RNA from the brome mosaic virus illustrates how cryoEM can detect inherent flexibility and how this relates to protein binding.
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.
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.
Protein synthesis of an RNA template can start by two different known mechanisms: cap-dependent translation initiation and cap-independent translation initiation. The latter is driven by RNA ...sequences called internal ribosome entry sites (IRESs) that are found in both viral RNAs and cellular mRNAs. The diverse mechanisms used by IRESs are reflected in their structural diversity, and this structural diversity challenges us to develop a cohesive model linking IRES function to structure. With more direct structural information available for the viral IRESs, data suggest an inverse correlation between the degree to which an IRES RNA can form a stable structure on its own and the number of factors that it requires to function. Lessons learned from the viral IRESs may help understand the cellular IRESs, although more structural data are needed before any strong links can be made.
Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell's RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that ...induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5' end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3' end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction's fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production. DOI: http://dx.doi.org/10.7554/eLife.01892.001.
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