Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

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Secondary Structural Model of Human MALAT1 Reveals Multiple Structure–Function Relationships

International Journal of Molecular Sciences ◽

10.3390/ijms20225610 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5610 ◽

Cited By ~ 10

Author(s):

Phillip J. McCown ◽

Matthew C. Wang ◽

Luc Jaeger ◽

Jessica A. Brown

Keyword(s):

Structure Function ◽

Noncoding Rna ◽

Structural Model ◽

Dynamic Structure ◽

Nucleotide Polymorphisms ◽

Rna Structures ◽

Rna Modifications ◽

Functional Roles ◽

Conserved Core

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an abundant nuclear-localized long noncoding RNA (lncRNA) that has significant roles in cancer. While the interacting partners and evolutionary sequence conservation of MALAT1 have been examined, much of the structure of MALAT1 is unknown. Here, we propose a hypothetical secondary structural model for 8425 nucleotides of human MALAT1 using three experimental datasets that probed RNA structures in vitro and in various human cell lines. Our model indicates that approximately half of human MALAT1 is structured, forming 194 helices, 13 pseudoknots, five structured tetraloops, nine structured internal loops, and 13 intramolecular long-range interactions that give rise to several multiway junctions. Evolutionary conservation and covariation analyses support 153 of 194 helices in 51 mammalian MALAT1 homologs and 42 of 194 helices in 53 vertebrate MALAT1 homologs, thereby identifying an evolutionarily conserved core that likely has important functional roles in mammals and vertebrates. Data mining revealed that RNA modifications, somatic cancer-associated mutations, and single-nucleotide polymorphisms may induce structural rearrangements that sequester or expose binding sites for several cancer-associated microRNAs. Our findings reveal new mechanistic leads into the roles of MALAT1 by identifying several intriguing structure–function relationships in which the dynamic structure of MALAT1 underlies its biological functions.

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Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523004113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7237-7242 ◽

Cited By ~ 43

Author(s):

Francesco Righetti ◽

Aaron M. Nuss ◽

Christian Twittenhoff ◽

Sascha Beele ◽

Kristina Urban ◽

...

Keyword(s):

Structural Changes ◽

Yersinia Pseudotuberculosis ◽

Dynamic Condition ◽

Rna Structures ◽

Bacterial Rna ◽

Different Temperatures ◽

Metabolic Transition ◽

Regulatory Potential ◽

Response To Temperature

RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37 °C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25 °C, 37 °C, and 42 °C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5′-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAs.

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Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

Nucleic Acids Research ◽

10.1093/nar/gkaa1053 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12436-12452 ◽

Cited By ~ 9

Author(s):

Ilaria Manfredonia ◽

Chandran Nithin ◽

Almudena Ponce-Salvatierra ◽

Pritha Ghosh ◽

Tomasz K Wirecki ◽

...

Keyword(s):

Secondary Structure ◽

Rna Structure ◽

Therapeutic Strategies ◽

Rna Structures ◽

Genome Wide ◽

High Sequence Conservation ◽

Infected Cells ◽

Rna Elements

Abstract SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

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Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3875 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3875-3891 ◽

Cited By ~ 46

Author(s):

Massimo Mallardo ◽

Sibylle Schleich ◽

Jacomine Krijnse Locker

Keyword(s):

Vaccinia Virus ◽

Rna Binding ◽

Rna Structures ◽

Viral Mrnas ◽

Virus Core ◽

Confocal Images ◽

Early Transcription ◽

Cytoplasmic Structures

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 μm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.

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A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Nucleic Acids Research ◽

10.1093/nar/gkaa1255 ◽

2021 ◽

Author(s):

Tycho Marinus ◽

Adam B Fessler ◽

Craig A Ogle ◽

Danny Incarnato

Keyword(s):

Rna Structure ◽

Structure Prediction ◽

Critical Role ◽

Living Cells ◽

Pathological Process ◽

Rna Structures ◽

Rna Molecules ◽

Derived Data

Abstract Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

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nextPARS: Parallel probing of RNA structures in Illumina

10.1101/174144 ◽

2017 ◽

Author(s):

Ester Saus ◽

Jesse Willis ◽

Leszek P. Pryszcz ◽

Heinz Himmelbauer ◽

Toni Gabaldón

Keyword(s):

Secondary Structures ◽

Structural Features ◽

Cellular Process ◽

Rna Structures ◽

Rna Molecules ◽

Comparable Accuracy

ABSTRACTRNA molecules play important roles in virtually every cellular process. These functions are often mediated through the adoption of specific structures that enable RNAs to interact with other molecules. Thus, determining the secondary structures of RNAs is central to understanding their function and evolution. In recent years several sequencing-based approaches have been developed that allow probing structural features of thousands of RNA molecules present in a sample. Here, we describe nextPARS, a novel Illumina-based implementation of in-vitro parallel probing of RNA structures. Our approach achieves comparable accuracy to previous implementations, while enabling higher throughput and sample multiplexing.

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Dual-gRNA Approach With Limited Off-Target Effect Corrects C9ORF72 Repeat Expansion In Vivo

10.21203/rs.3.rs-870138/v1 ◽

2021 ◽

Author(s):

Xuejiao Piao ◽

Dawei Meng ◽

Xue Zhang ◽

Qiang Song ◽

Hailong Lv ◽

...

Keyword(s):

Low Complexity ◽

Repeat Expansion ◽

Concept Design ◽

Rna Structures ◽

Dna And Rna ◽

Therapeutic Value ◽

Repeat Dna ◽

Target Effect

Abstract C9ORF72 GGGGCC repeat expansion is the most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia, which generates abnormal DNA and RNA structures and produces toxic proteins. Recently, efficacy of CRISPR/Cas9-mediated editing has been proven in treatment of disease. However, DNA low complexity surrounding C9ORF72 expansion increases the off-target risks. Here we provide a dual-gRNA design outside of the low complexity region which enables us to remove the repeat DNA in a ‘cutting-deletion-fusion’ manner with a high fusion efficiency (50%). Our dual-gRNA design limits off-target effect and does not significantly affect C9ORF72 expression. In neurons carrying patient C9ORF72 expansion, our approach removes the repeat DNA and corrects the RNA foci in vitro and in vivo. Therefore, we conclude that our proof-of-concept design correct C9ORF72 repeat expansion, which may have potential therapeutic value for the patients.

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RNA structure inference through chemical mapping after accidental or intentional mutations

10.1101/169953 ◽

2017 ◽

Author(s):

Clarence Y. Cheng ◽

Wipapat Kladwang ◽

Joseph Yesselman ◽

Rhiju Das

Keyword(s):

False Positive ◽

Structure Prediction ◽

Secondary Structure Prediction ◽

Dimethyl Sulfate ◽

Chemical Mapping ◽

Rna Structures ◽

Group I ◽

Egg Extract ◽

Special Cases

ABSTRACTDespite the critical roles RNA structures play in regulating gene expression, sequencing-based methods for experimentally determining RNA base pairs have remained inaccurate. Here, we describe a multidimensional chemical mapping method called M2-seq (mutate-and-map read out through next-generation sequencing) that takes advantage of sparsely mutated nucleotides to induce structural perturbations at partner nucleotides and then detects these events through dimethyl sulfate (DMS) probing and mutational profiling. In special cases, fortuitous errors introduced during DNA template preparation and RNA transcription are sufficient to give M2-seq helix signatures; these signals were previously overlooked or mistaken for correlated double DMS events. When mutations are enhanced through error-prone PCR, in vitro M2-seq experimentally resolves 33 of 68 helices in diverse structured RNAs including ribozyme domains, riboswitch aptamers, and viral RNA domains with a single false positive. These inferences do not require energy minimization algorithms and can be made by either direct visual inspection or by a new neural-net-inspired algorithm called M2-net. Measurements on the P4-P6 domain of the Tetrahymena group I ribozyme embedded in Xenopus egg extract demonstrate the ability of M2-seq to detect RNA helices in a complex biological environment.SIGNIFICANCE STATEMENTThe intricate structures of RNA molecules are crucial to their biological functions but have been difficult to accurately characterize. Multidimensional chemical mapping methods improve accuracy but have so far involved painstaking experiments and reliance on secondary structure prediction software. A methodology called M2-seq now lifts these limitations. Mechanistic studies clarify the origin of serendipitous M2-seq-like signals that were recently discovered but not correctly explained and also provide mutational strategies that enable robust M2-seq for new RNA transcripts. The method detects dozens of Watson-Crick helices across diverse RNA folds in vitro and within frog egg extract, with low false positive rate (< 5%). M2-seq opens a route to unbiased discovery of RNA structures in vitro and beyond.

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A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

10.1101/2020.08.31.274761 ◽

2020 ◽

Author(s):

Tycho Marinus ◽

Adam B. Fessler ◽

Craig A. Ogle ◽

Danny Incarnato

Keyword(s):

Rna Structure ◽

Structure Prediction ◽

Critical Role ◽

Living Cells ◽

Pathological Process ◽

Rna Structures ◽

Rna Molecules ◽

Derived Data

ABSTRACTDue to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art SHAPE reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

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