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

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.

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.

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.

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.

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.

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 . 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 . 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.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.