RNA Folding Research Articles

Genetic Variation Among Certain Fish Species in Terms of Evolution and Lineages

Three tests of phylogenetic including likelihood-joining tree, neighbour-joining tree, and minimum evolution tree have been used based on sox3 gene. Phylogenetic analysis was used to detect the genetic affinity and common ancestors for selected species that belong to the same or different families. This study showed the most appropriate methods for testing the genetic affinity among species and the methodology of each test according to the requirement of molecular applications. Secondary RNA predicted structure and minimum free energy were also included in this study because of their contribution to the detection of the orthologous gene and variance in RNA folding among species related to the different families. The genetic distance in the studied populations was calculated to know the most appropriate way to find out the genetic similarity among the studied species. The low distance-variance value of each group indicated significant genetic affinity among the species of the same family, this result is more consistent with the test of maximum-likelihood tree indicating the validity of this test to measure the genetic affinity among species that have common ancestors.

Regulation of the ThiM riboswitch is facilitated by the trapped structure formed during transcription of the wild-type sequence.

The ThiM riboswitch from Escherichia coli is a typical mRNA device that modulates downstream gene expression by sensing TPP. The helix-based RNA folding theory is used to investigate its detailed regulatory behaviors in cells. This RNA molecule is transcriptionally trapped in a state with the unstructured SD sequence in the absence of TPP, which induces downstream gene expression. As a key step to turn on gene expression, formation of this trapped state (the genetic ON state) highly depends on the co-transcriptional folding of its wild-type sequence. Instead of stabilities of the genetic ON and OFF states, the transcription rate, pause, and ligand levels are combined to affect the ThiM riboswitch-mediated gene regulation, which is consistent with a kinetic control model.

Coupling Between Production of Ribosomal RNA and Maturation: Just at the Beginning.

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35S/47S in yeast/human) is achieved by up to hundreds of RNA polymerase I (Pol I) enzymes simultaneously transcribing a single rRNA gene. In this review, we present recent advances in understanding the coupling between rRNA production and nascent rRNA folding. Mapping of the distribution of Pol I along ribosomal DNA at nucleotide resolution, using either native elongating transcript sequencing (NET-Seq) or crosslinking and analysis of cDNAs (CRAC), revealed frequent Pol I pausing, and CRAC results revealed a direct coupling between pausing and nascent RNA folding. High density of Pol I per gene imposes topological constraints that establish a defined pattern of polymerase distribution along the gene, with a persistent spacing between transcribing enzymes. RNA folding during transcription directly acts as an anti-pausing mechanism, implying that proper folding of the nascent rRNA favors elongation in vivo. Defects in co-transcriptional folding of rRNA are likely to induce Pol I pausing. We propose that premature termination of transcription, at defined positions, can control rRNA production in vivo.

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

The hexanucleotide repeat expansion GGGGCC [r(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G4C2)exp RNA gain- and loss-of-function mechanisms. The r(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

RR3DD: an RNA global structure-based RNA three-dimensional structural classification database

ABSTRACT The three-dimensional (3D) structure of RNA usually plays an important role in the recognition with RNA-binding protein. Along with the discovering of RNAs, several RNA databases are developed to study the functions of RNA based on sequence, secondary structure, local 3D structural motif and global structure. Based on RNA function and structure, different RNAs are classified and stored in SCOR and DARTS, respectively. The classification of RNA structures is useful in RNA structure prediction and function annotation. However, the SCOR and DARTS are not updated any more. In this study, we present an RNA classification database RR3DD based on RNA fold with the global 3D structural similarity. The RR3DD includes 13,601 RNA chains from PDB and mmCIF format structures which are classified into 780 RNA folds. The RNA chains from PDB and mmCIF format structures are aligned and clustered into 675 and 220 RNA folds, respectively. By analysing the RNA structure in RR3DD, we find that there are 11 clusters with more than 50 members. These clusters include rRNAs, riboswitches, tRNAs and so on. By mapping RR3DD into Rfam, we found that some RNAs without annotation by Rfam can be annotated through structural alignment. For example, we analysed tRNAs and found that tRNA were successfully grouped in RR3DD for which Rfam did not classify them into one family. Finally, we provide a web interface of RR3DD offering functions of browsing RR3DD, annotating RNA 3D structure and finding templates for RNA homology modelling.

Prediction of the Effects of Synonymous Variants on SARS-CoV-2 Genome

Background: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had led to a global pandemic since December 2019. SARS-CoV-2 is a single-stranded RNA virus, which mutates at a higher rate. Multiple studies had been done to identify and study nonsynonymous mutations, which change amino acid residues of SARS-CoV-2 proteins. On the other hand, there is little study on the effects of SARS-CoV-2 synonymous mutations. Although these mutations do not alter amino acids, some studies suggest that they may affect viral fitness. This study aims to predict the effect of synonymous mutations on the SARS-CoV-2 genome. Methods: A total of 30,229 SARS-CoV-2 genomic sequences were retrieved from Global Initiative on Sharing all Influenza Data (GISAID) database and aligned using MAFFT. Then, the mutations and their respective frequency were identified. A prediction of RNA secondary structures and their base pair probabilities was performed to study the effect of synonymous mutations on RNA structure and stability. Relative synonymous codon usage (RSCU) analysis was also performed to measure the codon usage bias (CUB) of SARS-CoV-2. Results: A total of 150 synonymous mutations were identified. The synonymous mutation identified with the highest frequency is C3037U mutation in the nsp3 of ORF1a, followed by C313U and C9286U mutation in nsp1 and nsp4 of ORF1a, respectively. Conclusion: Among the synonymous mutations identified, C913U mutation in ORF1a and C26735U in membrane (M) protein may affect RNA secondary structure, reducing the stability of RNA folding and possibly resulting in a higher translation rate. However, lab experiments are required to validate the results obtained from prediction analysis.

Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Visualizing formation of the active site in the mitochondrial ribosome.

Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.

AGO CLIP-based imputation of potent siRNA sequences targeting SARS-CoV-2 with antifibrotic miRNA-like activity

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2–8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-β signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online (http://clip.korea.ac.kr/covid19).

Nucleolar maturation of the human small subunit processome.

The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryo–electron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome’s impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA tool to target bacterial riboswitches and activate gene expression (rtRNA). The synthetic rtRNA is suitable for regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used small RNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic rtRNAs to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the rtRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

Vfold2D-MC: A Physics-Based Hybrid Model for Predicting RNA Secondary Structure Folding.

Accurate prediction of RNA structure and folding stability has a far-reaching impact on our understanding of RNA functions. Here we develop Vfold2D-MC, a new physics-based model, to predict RNA structure and folding thermodynamics from the sequence. The model employs virtual bond-based coarse-graining of RNA backbone conformation and generates RNA conformations through Monte Carlo sampling of the bond angles and torsional angles of the virtual bonds. Using a coarse-grained statistical potential derived from the known structures, we assign each conformation with a statistical weight. The weighted average over the conformational ensemble gives the entropy and free energy parameters for the hairpin, bulge, and internal loops, and multiway junctions. From the thermodynamic parameters, we predict RNA structures, melting curves, and structural changes from the sequence. Theory-experiment comparisons indicate that Vfold2D-MC not only gives improved structure predictions but also enables the interpretation of thermodynamic results for different RNA structures, including multibranched junctions. This new model sets a promising framework to treat more complicated RNA structures, such as pseudoknotted and intramolecular kissing loops, for which experimental thermodynamic parameters are often unavailable.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

Small Extracellular Vesicle Enrichment of a Retrotransposon-Derived Double-Stranded RNA: A Means to Avoid Autoinflammation?

Small extracellular vesicles (SEVs) such as exosomes are released by multiple cell types. Originally believed to be a mechanism for selectively removing unwanted cellular components, SEVs have received increased attention in recent years for their ability to mediate intercellular communication. Apart from proteins and lipids, SEVs contain RNAs, but how RNAs are selectively loaded into SEVs remains poorly understood. To address this question, we profiled SEV RNAs from mouse dendritic cells using RNA-Seq and identified a long noncoding RNA of retroviral origin, VL30, which is highly enriched (>200-fold) in SEVs compared to parental cells. Bioinformatic analysis revealed that exosome-enriched isoforms of VL30 RNA contain a repetitive 26-nucleotide motif. This repeated motif is itself efficiently incorporated into SEVs, suggesting the likelihood that it directly promotes SEV loading. RNA folding analyses indicate that the motif is likely to form a long double-stranded RNA hairpin and, consistent with this, its overexpression was associated with induction of a potent type I interferon response. Taken together, we propose that preferential loading into SEVs of the VL30 RNA containing this immunostimulatory motif enables cells to remove a potentially toxic RNA and avoid autoinflammation. In this way, the original notion of SEVs as a cellular garbage bin should not be entirely discounted.

An Approach to Transitioning Undergraduate Biochemistry Laboratory Courses Online

An Approach to Transitioning Undergraduate Biochemistry Laboratory Courses Online

Novel insights into the pervasive role of RNA structure in post-transcriptional regulation of gene expression in plants.

RNA folding is an intrinsic property of RNA that serves a key role in every step of post-transcriptional regulation of gene expression, from RNA maturation to translation in plants. Recent developments of genome-wide RNA structure profiling methods have transformed research in this area enabling focus to shift from individual molecules to the study of tens of thousands of RNAs. Here, we provide a comprehensive review of recent advances in the field. We discuss these new insights of RNA structure functionality within the context of post-transcriptional regulation including mRNA maturation, translation, and RNA degradation in plants. Notably, we also provide an overview of how plants exhibit different RNA structures in response to environmental changes.

A Mathematical Analysis of HDV Genotypes: From Molecules to Cells.

Hepatitis D virus (HDV) is classified according to eight genotypes. The various genotypes are included in the HDVdb database, where each HDV sequence is specified by its genotype. In this contribution, a mathematical analysis is performed on RNA sequences in HDVdb. The RNA folding predicted structures of the Genbank HDV genome sequences in HDVdb are classified according to their coarse-grain tree-graph representation. The analysis allows discarding in a simple and efficient way the vast majority of the sequences that exhibit a rod-like structure, which is important for the virus replication, to attempt to discover other biological functions by structure consideration. After the filtering, there remain only a small number of sequences that can be checked for their additional stem-loops besides the main one that is known to be responsible for virus replication. It is found that a few sequences contain an additional stem-loop that is responsible for RNA editing or other possible functions. These few sequences are grouped into two main classes, one that is well-known experimentally belonging to genotype 3 for patients from South America associated with RNA editing, and the other that is not known at present belonging to genotype 7 for patients from Cameroon. The possibility that another function besides virus replication reminiscent of the editing mechanism in HDV genotype 3 exists in HDV genotype 7 has not been explored before and is predicted by eigenvalue analysis. Finally, when comparing native and shuffled sequences, it is shown that HDV sequences belonging to all genotypes are accentuated in their mutational robustness and thermodynamic stability as compared to other viruses that were subjected to such an analysis.

OSTIR: open source translation initiation rate prediction.

SummaryTranslation of messenger RNAs into proteins by the ribosome is a fundamental step in gene expression. In bacteria, it is possible to accurately predict the rate of translation initiation from the sequence surrounding a gene’s start codon using thermodynamic models of RNA folding and ribosome binding. These predictions have applications in a range of fields, from systems biology studies that aim to understand and model bacterial physiology to synthetic biology studies that seek to reprogram bacterial cells. For example, metabolic engineers can design ribosome binding site (RBS) sequences to tune the expression of different enzymes in a pathway and thereby optimize the production of a desired chemical compound by cells. OSTIR (Open Source Translation Initiation Rates) is a Python package and command line tool for predicting translation initiation rates in bacteria.

Easy-Prime: a machine learning\u2013based prime editor design tool

Prime editing is a revolutionary genome-editing technology that can make a wide range of precise edits in DNA. However, designing highly efficient prime editors (PEs) remains challenging. We develop Easy-Prime, a machine learning–based program trained with multiple published data sources. Easy-Prime captures both known and novel features, such as RNA folding structure, and optimizes feature combinations to improve editing efficiency. We provide optimized PE design for installation of 89.5% of 152,351 GWAS variants. Easy-Prime is available both as a command line tool and an interactive PE design server at: http://easy-prime.cc/.

Cooperativity-Dependent Folding of Single-Stranded DNA

The folding of biological macromolecules is a fundamental process of which we lack a full comprehension. Mostly studied in proteins and RNA, single-stranded DNA (ssDNA) also folds, at physiological salt conditions, by forming non-specific secondary structures that are difficult to characterize with biophysical techniques. Here we present a helix-coil model for secondary structure formation, where ssDNA bases are organized in two different types of domains (compact and free). The model contains two parameters: the energy gain per base in a compact domain, $\epsilon$, and the cooperativity related to the interfacial energy between different domains, $\gamma$. We tested the ability of the model to quantify the formation of secondary structure in ssDNA molecules mechanically stretched with optical tweezers. The model reproduces the experimental force-extension curves in ssDNA of different molecular lengths and varying sodium and magnesium concentrations. Salt-correction effects for the energy of compact domains and the interfacial energy are found to be compatible with those of DNA hybridization. The model also predicts the folding free energy and the average size of domains at zero force, finding good agreement with secondary structure predictions by Mfold. We envision the model could be further extended to investigate native folding in RNA and proteins.