Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA.

RNA performs a wide range of functions regulated by its structure, dynamics, and often post-transcriptional modifications. While NMR is the leading method for understanding RNA structure and dynamics, it is currently limited by the inability to reduce spectral crowding by efficient segmental labeling. Furthermore, because of the challenging nature of RNA chemistry, the tools being developed to introduce site-specific modifications are increasingly complex and laborious. Here we use a previously designed Tgo DNA polymerase mutant to present SegModTeX - a versatile, one-pot, copy-and-paste approach to address these challenges. By precise, stepwise construction of a diverse set of RNA molecules, we demonstrate the technique to be superior to RNA polymerase driven and ligation methods owing to its substantially high yield, fidelity, and selectivity. We also show the technique to be useful for incorporating some fluorescent- and a wide range of other probes, which significantly extends the toolbox of RNA biology in general.

Extending the toolbox for RNA biology with SegModTeX: A polymerase-driven method for site-specific and segmental labeling of RNA.

RNA performs a wide range of functions regulated by its structure, dynamics, and often post-transcriptional modifications. While NMR is the leading method for understanding RNA structure and dynamics, it is currently limited by the inability to reduce spectral crowding by efficient segmental labeling. Furthermore, because of the challenging nature of RNA chemistry, the tools being developed to introduce site-specific modifications are increasingly complex and laborious. Here we use a previously designed Tgo DNA polymerase mutant to present SegModTeX - a versatile, one-pot, copy-and-paste approach to address these challenges. By precise, stepwise construction of a diverse set of RNA molecules, we demonstrate the technique to be superior to RNA polymerase driven and ligation methods owing to its substantially high yield, fidelity, and selectivity. We also show the technique to be useful for incorporating fluorescent- and a wide range of other probes, which significantly extends the toolbox of RNA biology in general.

A structure-based mechanism for displacement of the HEXIM adapter from 7SK small nuclear RNA.

Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between HEXIM and 7SK snRNA. In HIV-1, Tat binds to 7SK by displacing HEXIM. However, without the structure of the 7SK-HEXIM complex, the constraints that must be overcome for displacement remain unknown. Furthermore, while structure details of the TatNL4-3-7SK complex have been elucidated, it is unclear how subtypes with more HEXIM-like Tat sequences accomplish displacement. Here we report the structures of HEXIM, TatG, and TatFin arginine rich motifs in complex with the apical stemloop-1 of 7SK. While most interactions between 7SK with HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding - a feature that is then exploited by Tat to bind 7SK more efficiently.

Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome.

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome 1, 2 . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides 3-6 . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve 7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Alternate RNA Structures.

RNA molecules fold into complex three-dimensional structures that sample alternate conformations ranging from minor differences in tertiary structure dynamics to major differences in secondary structure. This allows them to form entirely different substructures with each population potentially giving rise to a distinct biological outcome. The substructures can be partitioned along an existing energy landscape given a particular static cellular cue or can be shifted in response to dynamic cues such as ligand binding. We review a few key examples of RNA molecules that sample alternate conformations and how these are capitalized on for control of critical regulatory functions.

HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.

The HIV Tat protein competes with the 7SK:HEXIM interaction to hijack pTEFb from 7SK snRNP and recruit it to the TAR motif on stalled viral transcripts. Here we solve structures of 7SK stemloop-1 and TAR in complex with Tat's RNA binding domain (RBD) to gain insights into this process. We find that 7SK is peppered with arginine sandwich motifs (ASM)-three classical and one with a pseudo configuration. Despite having similar RBDs, the presence of an additional arginine, R52, confers Tat the ability to remodel the pseudo configuration, required for HEXIM binding, into a classical sandwich, thus displacing HEXIM. Tat also uses R52 to remodel the TAR bulge into an ASM whose structure is identical to that of the remodeled ASM in 7SK. Together, our structures reveal a dual structural mimicry wherein viral Tat and TAR have co-opted structural motifs present in cellular HEXIM and 7SK for productive transcription of its genome.

An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation.

Many viruses bypass canonical cap-dependent translation in host cells by using internal ribosomal entry sites (IRESs) in their transcripts; IRESs hijack initiation factors for the assembly of initiation complexes. However, it is currently unknown how IRES RNAs recognize initiation factors that have no endogenous RNA binding partners; in a prominent example, the IRES of encephalomyocarditis virus (EMCV) interacts with the HEAT-1 domain of eukaryotic initiation factor 4G (eIF4G). Here we report the solution structure of the J-K region of this IRES and show that its stems are precisely organized to position protein-recognition bulges. This multisite interaction mechanism operates on an all-or-nothing principle in which all domains are required. This preorganization is accomplished by an 'adjuster module': a pentaloop motif that acts as a dual-sided docking station for base-pair receptors. Because subtle changes in the orientation abrogate protein capture, our study highlights how a viral RNA acquires affinity for a target protein.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing.

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA(Pro) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues--which are exactly complementary--for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA(Pro) reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies.

Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site-specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically (13) C-labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile (13) C and (15) N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

Determinants of Moloney murine leukemia virus Gag-Pol and genomic RNA proportions.

UNLABELLED: The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/Gag-Pol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of Gag-Pol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA. IMPORTANCE: Viruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.

HIV-1: packaging a shifty genome?

In this issue of Cell Host & Microbe, Chamanian et al. (2013) show that the frameshifting region in the HIV-1 genome influences the efficiency of genome packaging. This study may provide insights into mechanisms that constrain retroviruses into packaging only two copies of the genome during retroviral assembly.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding.

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag-Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5-10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag-Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography.

The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ(CD)](2), 132 nt, 42.8 kDa) using a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H-(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ(CD)](2) simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

Preformed protein-binding motifs in 7SK snRNA: structural and thermodynamic comparisons with retroviral TAR.

The 7SK small nuclear RNA is a highly conserved non-coding RNA that regulates transcriptional elongation. 7SK utilizes the HEXIM proteins to sequester the transcription factor P-TEFb by a mechanism similar to that used by retroviral TAR RNA to engage Tat and P-TEFb. Tat has also recently been shown to bind 7SK directly and recruit P-TEFb to TAR. We report here the solution structures of the free and arginine-bound forms of stem loop 4 of 7SK (7SK-SL4). Comparison of the 7SK-SL4 and TAR structures demonstrates the presence of a common arginine sandwich motif. However, arginine binding to 7SK-SL4 is mechanistically distinct and occurs via docking into a pre-organized pocket resulting in a 1000-fold increased affinity. Furthermore, whereas formation of the binding pocket in TAR requires a critical base-triple, hydrogen-bond formation between the equivalent bases in 7SK-SL4 is not essential and the pocket is stabilized solely by a pseudo base-triple platform. In addition, this theme of preformed protein binding motifs also extends into the pentaloop. The configuration of the loop suggests that 7SK-SL4 is poised to make ternary contacts with P-TEFb and HEXIM or Tat. These key differences between 7SK-SL4 and TAR present an opportunity to understand RNA structural adaptation and have implications for understanding differential interactions with Tat.

How retroviruses select their genomes.

As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein-RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.

Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription. Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled. Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein. Here we show that dimerization-induced register shifts in base pairing within the Psi-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 +/- 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide 'core encapsidation' segment of the MoMuLV Psi site reveals a network of interactions that promote sequence- and structure-specific binding by NC's single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus.

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.