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  1. Article: MRT-ModSeq - Rapid detection of RNA modifications with MarathonRT.

    Tavares, Rafael de Cesaris Araujo / Mahadeshwar, Gandhar / Wan, Han / Pyle, Anna Marie

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we ... ...

    Abstract Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT- ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2'-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.
    Language English
    Publishing date 2023-05-25
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.05.25.542276
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: MRT-ModSeq - Rapid Detection of RNA Modifications with MarathonRT.

    Araujo Tavares, Rafael de Cesaris / Mahadeshwar, Gandhar / Wan, Han / Pyle, Anna Marie

    Journal of molecular biology

    2023  Volume 435, Issue 22, Page(s) 168299

    Abstract: Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we ... ...

    Abstract Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT-ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2'-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.
    MeSH term(s) Mutation ; RNA Processing, Post-Transcriptional ; RNA, Ribosomal/chemistry ; RNA, Ribosomal/genetics ; Sequence Analysis, RNA/methods ; Humans
    Chemical Substances RNA, Ribosomal
    Language English
    Publishing date 2023-10-04
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2023.168299
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: RSCanner: rapid assessment and visualization of RNA structure content.

    Mahadeshwar, Gandhar / Tavares, Rafael de Cesaris Araujo / Wan, Han / Perry, Zion R / Pyle, Anna Marie

    Bioinformatics (Oxford, England)

    2023  Volume 39, Issue 3

    Abstract: Motivation: The increasing availability of RNA structural information that spans many kilobases of transcript sequence imposes a need for tools that can rapidly screen, identify, and prioritize structural modules of interest.: Results: We describe ... ...

    Abstract Motivation: The increasing availability of RNA structural information that spans many kilobases of transcript sequence imposes a need for tools that can rapidly screen, identify, and prioritize structural modules of interest.
    Results: We describe RNA Structural Content Scanner (RSCanner), an automated tool that scans RNA transcripts for regions that contain high levels of secondary structure and then classifies each region for its relative propensity to adopt stable or dynamic structures. RSCanner then generates an intuitive heatmap enabling users to rapidly pinpoint regions likely to contain a high or low density of discrete RNA structures, thereby informing downstream functional or structural investigation.
    Availability and implementation: RSCanner is freely available as both R script and R Markdown files, along with full documentation and test data (https://github.com/pylelab/RSCanner).
    MeSH term(s) RNA ; Software ; Protein Structure, Secondary ; Documentation ; Sequence Analysis, RNA
    Chemical Substances RNA (63231-63-0)
    Language English
    Publishing date 2023-03-21
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1422668-6
    ISSN 1367-4811 ; 1367-4803
    ISSN (online) 1367-4811
    ISSN 1367-4803
    DOI 10.1093/bioinformatics/btad111
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article: Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms.

    Huston, Nicholas C / Wan, Han / de Cesaris Araujo Tavares, Rafael / Wilen, Craig / Pyle, Anna Marie

    bioRxiv : the preprint server for biology

    2020  

    Abstract: SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as ... ...

    Abstract SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.
    Keywords covid19
    Language English
    Publishing date 2020-07-10
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2020.07.10.197079
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: G-quadruplex DNA structure is a positive regulator of

    Esain-Garcia, Isabel / Kirchner, Angie / Melidis, Larry / Tavares, Rafael de Cesaris Araujo / Dhir, Somdutta / Simeone, Angela / Yu, Zutao / Madden, Sarah K / Hermann, Regina / Tannahill, David / Balasubramanian, Shankar

    Proceedings of the National Academy of Sciences of the United States of America

    2024  Volume 121, Issue 7, Page(s) e2320240121

    Abstract: DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous ... ...

    Abstract DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human
    MeSH term(s) Humans ; DNA/metabolism ; G-Quadruplexes ; Gene Expression Regulation ; Promoter Regions, Genetic/genetics ; Transcription Factors/metabolism ; Proto-Oncogene Proteins c-myc/genetics
    Chemical Substances DNA (9007-49-2) ; Transcription Factors ; MYC protein, human ; Proto-Oncogene Proteins c-myc
    Language English
    Publishing date 2024-02-05
    Publishing country United States
    Document type Journal Article
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.2320240121
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms.

    Huston, Nicholas C / Wan, Han / Strine, Madison S / de Cesaris Araujo Tavares, Rafael / Wilen, Craig B / Pyle, Anna Marie

    Molecular cell

    2021  Volume 81, Issue 3, Page(s) 584–598.e5

    Abstract: Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as ...

    Abstract Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across β-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.
    MeSH term(s) COVID-19/genetics ; COVID-19/metabolism ; Cell Line, Tumor ; Genome, Viral ; Humans ; Nucleic Acid Conformation ; RNA, Viral/genetics ; RNA, Viral/metabolism ; Response Elements ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism
    Chemical Substances RNA, Viral
    Language English
    Publishing date 2021-01-01
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1415236-8
    ISSN 1097-4164 ; 1097-2765
    ISSN (online) 1097-4164
    ISSN 1097-2765
    DOI 10.1016/j.molcel.2020.12.041
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

    Tavares, Rafael de Cesaris Araujo / Mahadeshwar, Gandhar / Pyle, Anna Marie

    bioRxiv

    Abstract: SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about ... ...

    Abstract SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.
    Keywords covid19
    Publisher BioRxiv; WHO
    Document type Article ; Online
    DOI 10.1101/2020.07.06.190660
    Database COVID19

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  8. Article ; Online: The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

    Tavares, Rafael de Cesaris Araujo / Mahadeshwar, Gandhar / Pyle, Anna Marie

    bioRxiv

    Abstract: SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about ... ...

    Abstract SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.
    Keywords covid19
    Language English
    Publishing date 2020-07-07
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2020.07.06.190660
    Database COVID19

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  9. Article ; Online: The global and local distribution of RNA structure throughout the SARS-CoV-2 genome.

    Tavares, Rafael de Cesaris Araujo / Mahadeshwar, Gandhar / Wan, Han / Huston, Nicholas C / Pyle, Anna Marie

    Journal of virology

    2020  Volume 95, Issue 5

    Abstract: SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about ... ...

    Abstract SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ∼30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to predict regions of high base pair content across long genomes and to pinpoint hotspots of well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity for stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most structured viral RNAs in nature. Furthermore, our analysis suggests varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts, which given the technical difficulty of experimentally separating cellular mixtures of sgRNA from gRNA, is a unique advantage of our in-silico pipeline. The resulting findings provide a useful roadmap for planning focused empirical studies of SARS-CoV-2 RNA biology, and a preliminary guide for exploring potential SARS-CoV-2 RNA drug targets.
    Language English
    Publishing date 2020-12-02
    Publishing country United States
    Document type Journal Article
    ZDB-ID 80174-4
    ISSN 1098-5514 ; 0022-538X
    ISSN (online) 1098-5514
    ISSN 0022-538X
    DOI 10.1128/JVI.02190-20
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Comprehensive in-vivo secondary structure of SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

    Huston, Nicholas C / Wan, Han / Araujo Tavares, Rafael de Cesaris / Wilen, Craig B / Pyle, Anna Marie

    bioRxiv

    Abstract: SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as ... ...

    Abstract SARS-CoV-2 is the positive-sense RNA virus that causes COVID-19, a disease that has triggered a major human health and economic crisis. The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form stable RNA structures and yet, as much as 97% of its 30 kilobases have not been structurally explored in the context of a viral infection. Our limited knowledge of SARS-CoV-2 genomic architecture is a fundamental limitation to both our mechanistic understanding of coronavirus life cycle and the development of COVID-19 RNA-based therapeutics. Here, we apply a novel long amplicon strategy to determine for the first time the secondary structure of the SARS-CoV-2 RNA genome probed in infected cells. In addition to the conserved structural motifs at the viral termini, we report new structural features like a conformationally flexible programmed ribosomal frameshifting pseudoknot, and a host of novel RNA structures, each of which highlights the importance of studying viral structures in their native genomic context. Our in-depth structural analysis reveals extensive networks of well-folded RNA structures throughout Orf1ab and reveals new aspects of SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of RNA structures in SARS-CoV-2 shows that several features of its genomic structure are conserved across beta coronaviruses and we pinpoint individual regions of well-folded RNA structure that merit downstream functional analysis. The native, complete secondary structure of SAR-CoV-2 presented here is a roadmap that will facilitate focused studies on mechanisms of replication, translation and packaging, and guide the identification of new RNA drug targets against COVID-19.
    Keywords covid19
    Language English
    Publishing date 2020-07-10
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2020.07.10.197079
    Database COVID19

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