Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding

Ojha, Manju; Vogt, Jeff; Das, Naba Krishna; Redmond, Emily; Singh, Karndeep; Banna, Hasan Al; Sadat, Tasnia; Koirala, Deepak

doi:10.1073/pnas.2313677121

Cited by 3 publications

(1 citation statement)

References 62 publications

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“…Crystal structures have shown that a guanosine is extruded from a G-rich stretch in the PTE of the pea-enation mosaic virus (PEMV2), providing a structural basis for eIF4E recognition [ 207 ]. Similar studies in the saguaro cactus virus (SCV) revealed a flipped-out G residue ready to dock into the 5′ cap-binding pocket of eIF4E, highlighting 5′ cap mimicry by a 3′-CITE [ 208 ]. 3′-CITEs in T-shaped structures (TSSs) in TCV and cardamine chlorotic fleck virus RNAs can recycle post-termination ribosomes or ribosomal subunits directly to the 5′-end [ 209 , 210 ].…”

Section: 3′ End Elementsmentioning

confidence: 99%

Host-like RNA Elements Regulate Virus Translation

Khan,

Fox

2024

Viruses

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Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5′ or 3′ ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5′ cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This “thrifty” virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3′ end viral elements include 3′-cap-independent translation elements (3′-CITEs) and 3′-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

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Section: 3′ End Elementsmentioning

confidence: 99%

Host-like RNA Elements Regulate Virus Translation

Khan,

Fox

2024

Viruses

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Synthetic antibodies for accelerated RNA crystallography

DasGupta

2024

WIREs RNA

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RNA structure is crucial to a wide range of cellular processes. The intimate relationship between macromolecular structure and function necessitates the determination of high‐resolution structures of functional RNA molecules. X‐ray crystallography is the predominant technique used for macromolecular structure determination; however, solving RNA structures has been more challenging than their protein counterparts, as reflected in their poor representation in the Protein Data Bank (<1%). Antibody‐assisted RNA crystallography is a relatively new technique that promises to accelerate RNA structure determination by employing synthetic antibodies (Fabs) as crystallization chaperones that are specifically raised against target RNAs. Antibody chaperones facilitate the formation of ordered crystal lattices by minimizing RNA flexibility and replacing unfavorable RNA–RNA contacts with contacts between chaperone molecules. Atomic coordinates of these antibody fragments can also be used as search models to obtain phase information during structure determination. Antibody‐assisted RNA crystallography has enabled the structure determination of 15 unique RNA targets, including 11 in the last 6 years. In this review, I cover the historical development of antibody fragments as crystallization chaperones and their application to diverse RNA targets. I discuss how the first structures of antibody‐RNA complexes informed the design of second‐generation antibodies and led to the development of portable crystallization modules that have greatly reduced the uncertainties associated with RNA crystallography. Finally, I outline unexplored avenues that can increase the impact of this technology in structural biology research and discuss potential applications of antibodies as affinity reagents for interrogating RNA biology outside of their use in crystallography.This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein‐RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

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Synthetic anti-RNA antibody derivatives for RNA visualization in mammalian cells

Banna,

Berg,

Sadat

et al. 2024

Nucleic Acids Research

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Although antibody derivatives, such as Fabs and scFvs, have revolutionized the cellular imaging, quantification and tracking of proteins, analogous tools and strategies are unavailable for cellular RNA visualization. Here, we developed four synthetic anti-RNA scFv (sarabody) probes and their green fluorescent protein (GFP) fusions and demonstrated their potential to visualize RNA in live mammalian cells. We expressed these sarabodies and sarabody–GFP modules, purified them as soluble proteins, characterized their binding interactions with their corresponding epitopes and finally employed two of the four modules, sara1-GFP and sara1c-GFP, to visualize a target messenger RNA in live U2OS cells. Our current RNA imaging strategy is analogous to the existing MCP-MS2 system for RNA visualization, but additionally, our approach provides robust flexibility for developing target RNA-specific imaging modules, as epitope-specific probes can be selected from a library generated by diversifying the sarabody complementarity determining regions. While we continue to optimize these probes, develop new probes for various target RNAs and incorporate other fluorescence proteins like mCherry and HaloTag, our groundwork results demonstrated that these first-of-a-kind immunofluorescent probes will have tremendous potential for tracking mature RNAs and may aid in visualizing and quantifying many cellular processes as well as examining the spatiotemporal dynamics of various RNAs.

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Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding

Cited by 3 publications

References 62 publications

Host-like RNA Elements Regulate Virus Translation

Host-like RNA Elements Regulate Virus Translation

Synthetic antibodies for accelerated RNA crystallography

Synthetic anti-RNA antibody derivatives for RNA visualization in mammalian cells

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