RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has ...motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the ...genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.
Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates ...varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF.
The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic ...transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat–containing protein (LRPPRC)–deficient cells. Our findings elucidate LRPPRC’s role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.
Editor’s summary The secondary structure of mRNA plays crucial roles in gene expression. Moran et al . developed a chemical labeling approach (mitoDMS-MaPseq) and used a clustering algorithm to reveal the folding patterns of mitochondrial mRNA (mt-mRNA) in wild-type and LRPPRC-deficient cells. LRPPRC is a protein crucial for mt-mRNA maintenance and translation and acts as an mRNA holdase, influencing mRNA folding. Analyses of genome-wide and mt-mRNA–specific folding features revealed mRNA-programmed translational pausing and programmed ribosomal frameshifting. These findings establish a pivotal layer in mitochondrial gene expression, providing mt-mRNA folding maps for diverse physiological and pathological studies. —Di Jiang
INTRODUCTION Expression of the human mitochondrial genome (mtDNA) provides 13 essential protein subunits of the oxidative phosphorylation (OXPHOS) enzymatic complexes that catalyze aerobic energy transduction and support life. The mtDNA is a double-stranded 16.569-Kb molecule with heavy (H) and complementary light (L) strands. Transcription spans almost their entire length, producing two long polycistronic transcripts that are processed to yield 11 mature mRNAs, as two transcription units remain unprocessed as bicistronic elements containing overlapping and –1-shifted open reading frames (ORFs) encoding ATP8 and ATP6 or ND4L and ND4. The mtDNA also encodes the two ribosomal RNAs (12 S and 16 S rRNA) and 22 transfer RNAs required for synthesizing these proteins in mitochondrial ribosomes. Over the past five decades, the molecular machineries and mechanisms governing mtDNA transcription and mitochondrial messenger RNA (mt-mRNA) stability, processing, modification, and translation have been progressively characterized and remain the subject of intense investigations. However, our knowledge of the mt-mRNA folding landscape or structurome is very limited, which has hindered our mechanistic understanding of mtDNA gene expression and its regulation. RATIONALE Obtaining mRNA structure data within intact mitochondria is critical for understanding its biological context. To this goal, we have adapted an approach for the chemical probing of mRNA structures using dimethyl sulfate (DMS), which methylates the base-pairing faces of adenines and cytosines that are unpaired and accessible. Mitochondrial DMS mutational profiling with sequencing (mitoDMS-MaPseq) is a high-throughput, genome-wide RNA structure probing strategy that takes advantage of a high-fidelity and processive thermostable group II reverse transcriptase (TGIRT) enzyme that converts DMS modifications in the RNA to mismatches in the complementary DNA. DMS reactivity information is then used as a constraint in RNA folding algorithms based on thermodynamics to obtain highly accurate secondary structure models. RESULTS Having established a reproducible and accurate mitoDMS-MaPseq approach, we have investigated the structure of mt-mRNAs within functional human mitochondria. We used mitochondria isolated from wild-type cells and cells lacking the leucine-rich pentatricopeptide repeat–containing protein (LRPPRC), a pivotal regulator of mt-mRNA stability, polyadenylation, and translation. Our comparative analysis extended to in vitro synthesized and folded mt-mRNAs. Our findings elucidate LRPPRC’s role as a holdase that contributes to maintaining mt-mRNA folding and efficient translation. The mt-mRNA structural insights in wild-type mitochondria and metabolic labeling have unveiled potential mechanisms of gene expression. They include mRNA-programmed translational pausing to support the synthesis of COX1, a hydrophobic protein with multiple transmembrane domains, and a specific case of mRNA-programmed ribosome frameshifting (PRF) followed by termination-reinitiation events to coordinate translation of the two ORFs in the bicistronic ATP8/6 transcript. Furthermore, by using the clustering algorithm DREEM (detection of RNA folding ensembles using expectation-maximization), we have identified coexisting alternative conformations adopted by each transcript, capturing the dynamic nature of mt-mRNA folding in structural ensembles. CONCLUSION Our findings define the mt-mRNA folding landscape, offering insight into posttranscriptional regulation of mitochondrial gene expression. Additionally, we present a strategy that enables the investigation of mt-mRNA folding across cells and tissues and their role in regulating mitochondrial gene expression during development, diseases, and aging. The human mitochondrial mRNA folding landscape reveals mechanisms of gene expression. The mitoDMS-MaPseq workflow utilizes DMS modification of mt-RNAs, TGIRT-mediated mutagenesis, library preparation, deep sequencing, and conceptual analysis to probe mRNA secondary structures. The folding maps suggest gene expression mechanisms, including a potential programmed ribosome frameshifting in bicistronic ATP8/6 transcript translation. Mitoribosome, mitochondrial ribosome. Figure was created with BioRender.com
Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes ...alternative splicing to produce diverse protein products that include structural proteins and regulatory factors
. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements
. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages
, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized
alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis
that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.
Ribosome profiling data report on the distribution of translating ribosomes, at steady‐state, with codon‐level resolution. We present a robust method to extract codon translation rates and protein ...synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate‐reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.
Synopsis
Ribosome profiling experiments in wild‐type yeast and in mutants with altered tRNA levels illustrate that neither elongation rate nor translational efficiency is affected by tRNA abundance under physiological conditions.
A novel statistical model provides robust inference of codon translation rates and protein synthesis rates and hence better measures translation efficiency.
Codon translation rates have insignificant correlation with measures of codon bias.
Direct experimental manipulation of tRNA abundance does not affect elongation rates on affected codons or translation efficiency of overall genes.
Other sequence signals, such as mRNA structure and an initiation sequence motif, correlate to translation efficiency and may be causal determinants.
Ribosome profiling experiments in wild‐type yeast and in mutants with altered tRNA levels illustrate that neither elongation rate nor translational efficiency is affected by tRNA abundance under physiological conditions.
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide ...loss-of-function library in Saccaromyces cerevisiae as well as ∼78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
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► Comprehensive characterization of the stresses sensed by Hsf1 ► Characterization of a complex that targets ribosomes stalled at translation ► An autoregulatory loop regulates activity of the complex ► Discovery of a translation-stress signaling pathway from the ribosome to Hsf1
A ribosome-bound complex designated RQC associates with 60S ribosomal subunits containing stalled polypeptides to trigger their degradation.
Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite ...its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterize secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.
The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. ...Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.
A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a ...life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.
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