scholarly journals Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Methods ◽  
2016 ◽  
Vol 103 ◽  
pp. 34-48 ◽  
Author(s):  
Kyle E. Watters ◽  
Angela M Yu ◽  
Eric J. Strobel ◽  
Alex H. Settle ◽  
Julius B. Lucks
Keyword(s):  
Primer Extension ◽  
Rna Structures

scholarly journals Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

2015 ◽  
Author(s):  
Kyle E Watters ◽  
Angela M Yu ◽  
Eric J Strobel ◽  
Alex H Settle ◽  
Julius Lucks
Keyword(s):  
Primer Extension ◽  
Computational Techniques ◽  
Rna Structures ◽  
Cellular Functions ◽  
Rna Molecules ◽  
Cellular Defense ◽  
Nucleotide Resolution ◽  
And Function

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

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

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.

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

2020 ◽  
Vol 48 (22) ◽  
pp. 12436-12452 ◽  
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.

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

2001 ◽  
Vol 12 (12) ◽  
pp. 3875-3891 ◽  
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.

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

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.

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

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.

scholarly journals An Exclusively Nuclear RNA-Binding Protein Affects Asymmetric Localization of ASH1 mRNA and Ash1p in Yeast

2001 ◽  
Vol 153 (2) ◽  
pp. 307-318 ◽  
Author(s):  
Roy M. Long ◽  
Wei Gu ◽  
Xiuhua Meng ◽  
Graydon Gonsalvez ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Yeast Cells ◽  
Rna Structures ◽  
Nuclear Factors ◽  
Mother Cells ◽  
Ash1 Mrna

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3′-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3′-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

scholarly journals A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation

2019 ◽  
Vol 47 (17) ◽  
pp. 9329-9342 ◽  
Author(s):  
Chaminda D Gunawardene ◽  
Laura R Newburn ◽  
K Andrew White
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Degradation Products ◽  
Rna Degradation ◽  
Viral Rna ◽  
Tobacco Necrosis Virus ◽  
Rna Structures ◽  
Necrosis Virus

Abstract Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

scholarly journals Community science designed ribosomes with beneficial phenotypes

2021 ◽  
Author(s):  
Antje Kruger ◽  
Andrew M Watkins ◽  
Roger Wellington-Oguri ◽  
Jonathan Romano ◽  
Camila Kofman ◽  
...  
Keyword(s):  
Video Game ◽  
Rational Design ◽  
Large Scale ◽  
Rna Structures ◽  
3D Design ◽  
Design Build ◽  
Community Science ◽  
Rrna Sequences

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

scholarly journals RNA G-quadruplex structures exist and function in vivo

2019 ◽  
Author(s):  
Xiaofei Yang ◽  
Jitender Cheema ◽  
Yueying Zhang ◽  
Hongjing Deng ◽  
Susan Duncan ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Rna Structures ◽  
Specific Sequence ◽  
Form Complex ◽  
Folding Potential ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
And Function

AbstractGuanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

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