Local RNA Structure Research Articles

Alternative production of pro-death Bax∆2 protein via ribosomal frameshift in Alzheimer’s disease

Pro-death Bax family member, Bax∆2, forms protein aggregates in Alzheimer’s disease neurons and causes stress granule-mediated neuronal death. Production of Bax∆2 originated from two events: alternative splicing of Bax exon 2 and a microsatellite mutation (a deletion from poly guanines, G8 to G7). Each event alone leads to a reading frameshift and premature termination. While Bax exon 2 alternative splicing is common in Alzheimer’s brains, the G7 mutation is not, which is inconsistent with the high Bax∆2 protein levels detected in clinical samples. Here, we report an alternative mechanism to produce Bax∆2 protein in the absence of the G7 mutation. Using dual-tag systems, we showed that a ribosomal frameshift (RFS) can compensate the lack of G7 mutation in translating Bax∆2 protein. Intriguingly, the microsatellite poly G repeat is neither essential nor the site for the RFS. However, disruption of the poly G sequence impaired the RFS, potentially due to alteration of the local RNA structure. Using immunoprecipitation-mass spectrometry, we pinpointed the RFS site at 15 base pairs upstream of the microsatellite. These results uncover an alternative mechanism for generating functional Bax∆2-subfamily isoforms, highlighting the production plasticity of Bax family isoforms and unveiling potential new therapeutic targets for Alzheimer’s disease.

Evaluation of the effect of RNA secondary structure on Cas13d-mediated target RNA cleavage

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the SARS-CoV-2 target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. Comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structure than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

The double-stranded microRNA precursor.

MicroRNAs (miRNAs) are generated from stem-loop-structured double-stranded RNA precursors by the consecutive action of the two RNase III-type endoribonuclease Drosha and Dicer. However, such structures are very common on cellular transcripts and specific features have evolved that guide and regulate processing of stem-loop-structured hairpins into mature and functional miRNAs. These features include sequence motifs and local RNA structures but also trans-acting factors such as RNA binding proteins. The menu of features required for miRNA biogenesis is summarized in this review.

Cooperation and Competition of RNA Secondary Structure and RNA-Protein Interactions in the Regulation of Alternative Splicing.

The regulation of alternative splicing in eukaryotic cells is carried out through the coordinated action of a large number of factors, including RNA-binding proteins and RNA structure. The RNA structure influences alternative splicing by blocking cis-regulatory elements, or bringing them closer or farther apart. In combination with RNA-binding proteins, it generates transcript conformations that help to achieve the necessary splicing outcome. However, the binding of regulatory proteins depends on RNA structure and, vice versa, the formation of RNA structure depends on the interaction with regulators. Therefore, RNA structure and RNA-binding proteins are inseparable components of common regulatory mechanisms. This review highlights examples of alternative splicing regulation by RNA-binding proteins, the regulation through local and long-range RNA structures, as well as how these elements work together, cooperate, and compete.

Influence of Mg2+ Distribution on the Stability of Folded States of the Twister Ribozyme Revealed Using Grand Canonical Monte Carlo and Generative Deep Learning Enhanced Sampling.

Metal ions, particularly magnesium ions (Mg2+), play a role in stabilizing the tertiary structures of RNA molecules. Theoretical models and experimental techniques show that metal ions can change RNA dynamics and how it transitions through different stages of folding. However, the specific ways in which metal ions contribute to the formation and stabilization of RNA's tertiary structure are not fully understood at the atomic level. Here, we combined oscillating excess chemical potential Grand Canonical Monte Carlo (GCMC) and metadynamics to bias toward the sampling of unfolded states using reaction coordinates generated by machine learning allowing for examination of Mg2+-RNA interactions that contribute to stabilizing folded states of the pseudoknot found in the Twister ribozyme. GCMC is used to sample diverse ion distributions around the RNA with deep learning applied to iteratively generate system-specific reaction coordinates to maximize conformational sampling during metadynamics simulations. Results from 6 μs simulations performed on 9 individual systems indicate that Mg2+ ions play a crucial role in stabilizing the three-dimensional (3D) structure of the RNA by stabilizing specific interactions of phosphate groups or phosphate groups and bases of neighboring nucleotides. While many phosphates are accessible to interactions with Mg2+, it is observed that multiple, specific interactions are required to sample conformations close to the folded state; coordination of Mg2+ at individual specific sites facilitates sampling of folded conformations though unfolding ultimately occurs. It is only when multiple specific interactions occur, including the presence of specific inner-shell cation interactions linking two nucleotides, that conformations close to the folded state are stable. While many of the identified Mg2+ interactions are observed in the X-ray crystal structure of Twister, the present study suggests two new Mg2+ ion sites in the Twister ribozyme that contribute to stabilization. In addition, specific interactions with Mg2+ are observed that destabilize the local RNA structure, a process that may facilitate the folding of RNA into its correct structure.

EIF4A1-dependent mRNAs employ purine-rich 5'UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation.

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

C-to-U RNA deamination is the driving force accelerating SARS-CoV-2 evolution.

Understanding the molecular mechanism underlying the rampant mutation of SARS-CoV-2 would help us control the COVID-19 pandemic. The APOBEC-mediated C-to-U deamination is a major mutation type in the SARS-CoV-2 genome. However, it is unclear whether the novel mutation rate u is higher for C-to-U than for other mutation types, and what the detailed driving force is. By analyzing the time course SARS-CoV-2 global population data, we found that C-to-U has the highest novel mutation rate u among all mutation types and that this u is still increasing with time (du/dt > 0). Novel C-to-U events, rather than other mutation types, have a preference over particular genomic regions. A less local RNA structure is correlated with a high novel C-to-U mutation rate. A cascade model nicely explains the du/dt > 0 for C-to-U deamination. In SARS-CoV-2, the RNA structure serves as the molecular basis of the extremely high and continuously accelerating C-to-U deamination rate. This mechanism is the driving force of the mutation, adaptation, and evolution of SARS-CoV-2. Our findings help us understand the dynamic evolution of the virus mutation rate.

Probing RNA Structures and Interactions Using Fluorescence Lifetime Analyses.

Structural analyses of large, complex noncoding RNAs continue to lag behind their rapid discovery and functional descriptions. Site-specifically incorporated, minimally invasive fluorescent probes such as 2-aminopurine (2AP) and pyrrolo-cytosine (PyC) have provided essential complementary information about local RNA structure, conformational dynamics, and interactions. Here I describe a protocol that benchmarks and correlates local RNA conformations with their respective fluorescence lifetimes, as a general technique that confers key advantages over fluorescence intensity-based methods. The observation that fluorescence lifetimes are more sensitive to local structures than sequence contexts suggests broad utility across diverse RNA and ribonucleoprotein systems.

Global 5'-UTR RNA structure regulates translation of a SERPINA1 mRNA.

SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5′-untranslated region (5′-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5′-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5′-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5′-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5′-UTR structure. However, those mutations that disrupted the helix that anchors the 5′-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5′-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5′-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.

Silent codon positions in the A-rich HIV RNA genome that do not easily become A: Restrictions imposed by the RNA sequence and structure.

There is a strong evolutionary tendency of the human immunodeficiency virus (HIV) to accumulate A nucleotides in its RNA genome, resulting in a mere 40 per cent A count. This A bias is especially dominant for the so-called silent codon positions where any nucleotide can be present without changing the encoded protein. However, particular silent codon positions in HIV RNA refrain from becoming A, which became apparent upon genome analysis of many virus isolates. We analyzed these 'noA' genome positions to reveal the underlying reason for their inability to facilitate the A nucleotide. We propose that local RNA structure requirements can explain the absence of A at these sites. Thus, noA sites may be prominently involved in the correct folding of the viral RNA. Turning things around, the presence of multiple clustered noA sites may reveal the presence of important sequence and/or structural elements in the HIV RNA genome.

Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria.

In land plant mitochondria, C-to-U RNA editing converts cytidines into uridines at highly specific RNA positions called editing sites. This editing step is essential for the correct functioning of mitochondrial proteins. When using sequence homology information, edited positions can be computationally predicted with high precision. However, predictions based on the sequence contexts of such edited positions often result in lower precision, which is limiting further advances on novel genetic engineering techniques for RNA regulation. Here, a deep convolutional neural network called Deepred-Mt is proposed. It predicts C-to-U editing events based on the 40 nucleotides flanking a given cytidine. Unlike existing methods, Deepred-Mt was optimized by using editing extent information, novel strategies of data augmentation, and a large-scale training dataset, constructed with deep RNA sequencing data of 21 plant mitochondrial genomes. In comparison to predictive methods based on sequence homology, Deepred-Mt attains significantly better predictive performance, in terms of average precision as well as F1 score. In addition, our approach is able to recognize well-known sequence motifs linked to RNA editing, and shows that the local RNA structure surrounding editing sites may be a relevant factor regulating their editing. These results demonstrate that Deepred-Mt is an effective tool for predicting C-to-U RNA editing in plant mitochondria. Source code, datasets, and detailed use cases are freely available at https://github.com/aedera/deepredmt.

The ‘hidden side’ of spin labelled oligonucleotides: Molecular dynamics study focusing on the EPR-silent components of base pairing

Nitroxide labels are combined with nucleic acid structures and are studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2′-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence- dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labelled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabelled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies.

Amino Acid Composition in Various Types of Nucleic Acid-Binding Proteins.

Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

Transcriptome-wide analysis reveals spatial correlation between N6-methyladenosine and binding sites of microRNAs and RNA-binding proteins

N6-methyladenosine (m6A), the most prevalent epitranscriptomic modification in eukaryotes, is enriched in 3′-untranslated regions (3′UTRs) of mRNAs. As 3′UTRs are major binding sites of RNA-binding proteins (RBPs) and microRNAs (miRNAs), m6A-dependent local RNA structure change may alter the accessibility of RBPs and miRNAs to their target sites and regulate mRNA function. Using a human transcriptome-wide computational analysis to investigate the relation between m6A, RBPs and miRNAs, we find a strong positive correlation between number of m6A sites, miRNAs and RBPs binding to mRNAs, suggesting m6A-modified mRNAs are more targeted by miRNAs and RBPs. Moreover, m6A sites are located proximally to miRNA target sites and binding sites of multiple RBPs. Further, miRNA target sites and RBP-binding sites located close to each other are also located proximally to m6A. This study indicates three-way interplay between m6A, microRNA and RBP binding, suggesting the influence of mRNA modifications on the miRNA and RBP interactomes.

SHAPE Profiling to Probe Group II Intron Conformational Dynamics During Splicing.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a widely used technique for studying the structure and function of RNA molecules. It characterizes the flexibility of single nucleotides in the context of the local RNA structure. Here we describe the application of SHAPE-MaP (mutational profiling) to study different conformational states of the group II intron during the self-splicing reaction.

Effect of methylation of adenine N6 on kink turn structure depends on location

ABSTRACT N6-methyladenine is the most common covalent modification in cellular RNA species, with demonstrated functional consequences. At the molecular level this methylation could alter local RNA structure, and/or modulate the binding of specific proteins. We have previously shown that trans-Hoogsteen-sugar (sheared) A:G base pairs can be completely disrupted by methylation, and that this occurs in a sub-set ofD/D k-turn structures. In this work we have investigated to what extent sequence context affects the severity with which inclusion of N6-methyladenine into different A:G base pairs of a standard k-turn affects RNA folding and L7Ae protein binding. We find that local sequence has a major influence, ranging from complete absence of folding and protein binding to a relatively mild effect. We have determined the crystal structure of one of these species both free and protein-bound, showing the environment of the methyl group and the way the modification is accommodated into the k-turn structure.

Towards Long-Range RNA Structure Prediction in Eukaryotic Genes.

The ability to form an intramolecular structure plays a fundamental role in eukaryotic RNA biogenesis. Proximate regions in the primary transcripts fold into a local secondary structure, which is then hierarchically assembled into a tertiary structure that is stabilized by RNA-binding proteins and long-range intramolecular base pairings. While the local RNA structure can be predicted reasonably well for short sequences, long-range structure at the scale of eukaryotic genes remains problematic from the computational standpoint. The aim of this review is to list functional examples of long-range RNA structures, to summarize current comparative methods of structure prediction, and to highlight their advances and limitations in the context of long-range RNA structures. Most comparative methods implement the “first-align-then-fold” principle, i.e., they operate on multiple sequence alignments, while functional RNA structures often reside in non-conserved parts of the primary transcripts. The opposite “first-fold-then-align” approach is currently explored to a much lesser extent. Developing novel methods in both directions will improve the performance of comparative RNA structure analysis and help discover novel long-range structures, their higher-order organization, and RNA–RNA interactions across the transcriptome.

Polymorphism studies on microRNA targetome of thalassemia.

Thalassemia is one of the most prevalent hemoglobin disorders. It is caused by the decreased or absent synthesis of one globin chain that leads to moderate to severe hemolytic anemia in clinical complications. Some genetic factors cause these phenotypic variations by the alteration of gene expression. MicroRNAs (miRNAs) are post-transcriptional regulators in gene expression. Therefore, variations in 3'-untranslated region (3'-UTR) of target genes may affect gene expression. It is of interest to evaluate the impact of noncoding SNPs in thalassemia related genes on miRNA: mRNA interactions in the severity of thalassemia. Polymorphisms that alter miRNA: mRNA interactions were predicted using PolymiRTS and Mirsnpscore tools. Then, the effect of predicted target SNPs on thermodynamic stability, local RNA structure and regulatory elements was investigated using RNAhybrid, RNAsnp and RegulomeDB, respectively. The molecular functions and the Biological process of candidate genes were extracted and interaction network was created. Forty-six SNPs were predicted to affect 188 miRNA interactions. These results suggest that 3'-UTR SNP may affect gene expression and cause phenotypic variation in thalassemia patients.

The Evolution of Substrate Specificity by tRNA Modification Enzymes.

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability. Whichever the case, the overall importance of modifications is highlighted by both their evolutionary conservation and the fact that organisms use a substantial portion of their genomes to encode modification enzymes, far exceeding what is needed for the de novo synthesis of the canonical nucleotides themselves [2]. Although some modifications occur at exactly the same nucleotide position in tRNAs from the three domains of life, many can be found at various positions in a particular tRNA and their location may vary between and within different tRNAs. With this wild array of chemical diversity and substrate specificities, one of the big challenges in the tRNA modification field has been to better understand at a molecular level the modes of substrate recognition by the different modification enzymes; in this realm RNA binding rests at the heart of the problem. This chapter will focus on several examples of modification enzymes where their mode of RNA binding is well understood; from these, we will try to draw general conclusions and highlight growing themes that may be applicable to the RNA modification field at large.

Cover Caption: Functions of RNA Modifications

Journal of Integrative Plant BiologyVolume 58, Issue 10 p. C1-C1 Cover PictureFree Access Cover Caption: Functions of RNA Modifications First published: 02 October 2016 https://doi.org/10.1111/jipb.12412AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract More than 100 RNA modifications are known in animals and plants. In this issue, Burgess et al. (822–835) provide a general review of known biological functions of RNA modifications, and enzymes mediating these modifications in plants. The cover image summarizes how RNA modifications can add another layer of regulation to the transcriptome by remodeling local RNA structure. Volume58, Issue10October 2016Pages C1-C1 RelatedInformation