Structure-guided aminoacylation and assembly of chimeric RNAs.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messenger RNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

The SARS-CoV-2 Programmed -1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography.

The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

Synthetic Antibody Binding to a Preorganized RNA Domain of Hepatitis C Virus Internal Ribosome Entry Site Inhibits Translation.

Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). We used these Fabs as crystallization chaperones and determined the high-resolution crystal structures of JIIIabc-HCV2 and -HCV3 complexes at 1.81 Å and 2.75 Å resolution respectively, revealing an antiparallel four-way junction with the IIIa and IIIc subdomains brought together through tertiary interactions. The RNA conformation observed in the structures supports the structural model for this region derived from cryo-EM data for the HCV IRES-40S ribosome complex, suggesting that the tertiary fold of the RNA preorganizes the domain for interactions with the 40S ribosome. Strikingly, both Fabs and the ribosomal protein eS27 not only interact with a common subset of nucleotides within the JIIIabc but also use physiochemically similar sets of protein residues to do so, suggesting that the RNA surface is well-suited for interactions with proteins, perhaps analogous to the "hot spot" concept elaborated for protein-protein interactions. Using a rabbit reticulocyte lysate-based translation assay with a bicistronic reporter construct, we further demonstrated that Fabs HCV2 and HCV3 specifically inhibit the HCV IRES-directed translation, implicating disruption of the JIIIabc-ribosome interaction as a potential therapeutic strategy against HCV.

The effect of muscle glycogen phosphorylase (Pygm) knockdown on zebrafish morphology.

Muscle glycogen phosphorylase (PYGM) is a key enzyme in the first step of glycogenolysis. Mutation in the PYGM gene leads to autosomal recessive McArdle disease. Patients suffer from exercise intolerance with premature fatigue, muscle cramps and myalgia due to lack of available glucose in muscles. So far, no efficient treatment has been found. The zebrafish has many experimental advantages, and was successfully implemented as an animal model of human myopathies. Since zebrafish skeletal muscles share high similarity with human skeletal muscles, it is our animal of choice to investigate the impact of Pygm knockdown on skeletal muscle tissue. The two forms of the zebrafish enzyme, Pygma and Pygmb, share more than 80% amino acid sequence identity with human PYGM. We show that the Pygm level varies at both the mRNA and protein level in distinct stages of zebrafish development, which is correlated with glycogen level. The Pygm distribution in muscles varies from dispersed to highly organized at 72 hpf. The pygma and pygmb morpholino knockdown resulted in a reduced Pygm level in zebrafish morphants, which exhibited altered, disintegrated muscle structure and accumulation of glycogen granules in the subsarcolemmal region. Thus, lowering the Pygm level in zebrafish larvae leads to an elevated glycogen level and to morphological muscle changes mimicking the symptoms of human McArdle disease. The zebrafish model of this human disease might contribute to further understanding of its molecular mechanisms and to the development of appropriate treatment.

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function.

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans.

Expression of vascular endothelial growth factor mRNA in human leiomyomas.

AIM: Growth factors play a major role in the pathogenesis of uterine myomas. The aim of this study was to evaluate vascular endothelial growth factor (VEGF) mRNA expression during leiomyoma growth at different phases of the menstrual cycle with RT-PCR. METHOD: We studied 56 patients: 43 with myomas, 13 with healthy myometriums. In patients with myomas (secretory phase), VEGF expression was 2.82 times higher than in control patients (p < 0.05). In patients with myomas (phase I), VEGF expression was 2.53 times higher (p < 0.05) than in control patients. For all patients with myomas, those who were in menopause had 1.52 times higher VEGF expression than those who menstruated. For patients with healthy myometriums, those who were in menopause had 1.97 times higher VEGF expression than those who menstruated. A comparison of all the patients in menopause revealed that VEGF expression was 2.03-fold higher in those with myomas than in those with healthy myometriums. CONCLUSION: We observed the highest VEGF mRNA expression in women with myomas who were in menopause. Among menstruating patients, VEGF expression was significantly higher in those with myomas compared to those with a healthy myometrium. This suggested that VEGF may play a significant role in the pathogenesis of uterine myomas.