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  1. Article ; Online: FLARE

    Eric Kofman / Brian Yee / Hugo C. Medina-Munoz / Gene W. Yeo

    BMC Bioinformatics, Vol 24, Iss 1, Pp 1-

    a fast and flexible workflow for identifying RNA editing foci

    2023  Volume 15

    Abstract: Abstract Background Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base ... ...

    Abstract Abstract Background Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors. Results We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing. Conclusions FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE .
    Keywords RNA editing ; Clustering ; Statistical ; Modeling ; Computer applications to medicine. Medical informatics ; R858-859.7 ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2023-10-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Non-microRNA binding competitively inhibits LIN28 regulation

    Frederick E. Tan / Shashank Sathe / Emily C. Wheeler / Gene W. Yeo

    Cell Reports, Vol 36, Iss 6, Pp 109517- (2021)

    2021  

    Abstract: Summary: RNA binding protein (RBP) expression is finite. For RBPs that are vastly outnumbered by their potential target sites, a simple competition for binding can set the magnitude of post-transcriptional control. Here, we show that LIN28, best known ... ...

    Abstract Summary: RNA binding protein (RBP) expression is finite. For RBPs that are vastly outnumbered by their potential target sites, a simple competition for binding can set the magnitude of post-transcriptional control. Here, we show that LIN28, best known for its direct regulation of let-7 miRNA biogenesis, is also indirectly regulated by its widespread binding of non-miRNA transcripts. Approximately 99% of LIN28 binding sites are found on non-miRNA transcripts, like protein coding and ribosomal RNAs. These sites are bound specifically and strongly, but they do not appear to mediate direct post-transcriptional regulation. Instead, non-miRNA sites act to sequester LIN28 protein and effectively change its functional availability, thus impeding the regulation of let-7 in cells. Together, these data show that the binding properties of the transcriptome broadly influence the ability of an RBP to mediate changes in RNA metabolism and gene expression.
    Keywords RNA binding proteins ; post-transcriptional regulation ; microRNA ; non-miRNA ; ribosome occupancy ; competitive inhibition ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2021-08-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Crosstalk between CRISPR-Cas9 and the human transcriptome

    Aaron A. Smargon / Assael A. Madrigal / Brian A. Yee / Kevin D. Dong / Jasmine R. Mueller / Gene W. Yeo

    Nature Communications, Vol 13, Iss 1, Pp 1-

    2022  Volume 8

    Abstract: The off-target effects of CRISPR-Cas9 are thought to be mediated by its cognate guide RNA. Here the authors show that Cas9 independently interacts with the human transcriptome, correlating with elevated RNA editing even under guide RNA co-expression. ...

    Abstract The off-target effects of CRISPR-Cas9 are thought to be mediated by its cognate guide RNA. Here the authors show that Cas9 independently interacts with the human transcriptome, correlating with elevated RNA editing even under guide RNA co-expression.
    Keywords Science ; Q
    Language English
    Publishing date 2022-03-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: The ViReflow pipeline enables user friendly large scale viral consensus genome reconstruction

    Niema Moshiri / Kathleen M. Fisch / Amanda Birmingham / Peter DeHoff / Gene W. Yeo / Kristen Jepsen / Louise C. Laurent / Rob Knight

    Scientific Reports, Vol 12, Iss 1, Pp 1-

    2022  Volume 6

    Abstract: Abstract Throughout the COVID-19 pandemic, massive sequencing and data sharing efforts enabled the real-time surveillance of novel SARS-CoV-2 strains throughout the world, the results of which provided public health officials with actionable information ... ...

    Abstract Abstract Throughout the COVID-19 pandemic, massive sequencing and data sharing efforts enabled the real-time surveillance of novel SARS-CoV-2 strains throughout the world, the results of which provided public health officials with actionable information to prevent the spread of the virus. However, with great sequencing comes great computation, and while cloud computing platforms bring high-performance computing directly into the hands of all who seek it, optimal design and configuration of a cloud compute cluster requires significant system administration expertise. We developed ViReflow, a user-friendly viral consensus sequence reconstruction pipeline enabling rapid analysis of viral sequence datasets leveraging Amazon Web Services (AWS) cloud compute resources and the Reflow system. ViReflow was developed specifically in response to the COVID-19 pandemic, but it is general to any viral pathogen. Importantly, when utilized with sufficient compute resources, ViReflow can trim, map, call variants, and call consensus sequences from amplicon sequence data from 1000 SARS-CoV-2 samples at 1000X depth in < 10 min, with no user intervention. ViReflow’s simplicity, flexibility, and scalability make it an ideal tool for viral molecular epidemiological efforts.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2022-03-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins

    Meredith Corley / Ryan A. Flynn / Steven M. Blue / Brian A. Yee / Howard Y. Chang / Gene W. Yeo

    STAR Protocols, Vol 2, Iss 3, Pp 100762- (2021)

    2021  

    Abstract: Summary: Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE ( ... ...

    Abstract Summary: Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE.For complete details on the use and execution of these protocols, please refer to Corley et al. (2020).
    Keywords Genomics ; RNAseq ; Molecular Biology ; Antibody ; Science (General) ; Q1-390
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics

    Sebastian Markmiller / Amit Fulzele / Reneé Higgins / Marilyn Leonard / Gene W. Yeo / Eric J. Bennett

    Cell Reports, Vol 27, Iss 5, Pp 1356-1363.e

    2019  Volume 3

    Abstract: Summary: Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway ... ...

    Abstract Summary: Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics. We show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Critically, inhibiting neither UAE nor NAE significantly affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Using antibodies with varying preference for free ubiquitin or polyubiquitin and fluorescently tagged ubiquitin variants in combination with UAE inhibition, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions. : Protein ubiquitylation has been implicated in pathways by which cellular stress induces the formation of stress granules (SGs) and affects protein homeostasis through the ubiquitin proteasome system. Markmiller et al. show that ubiquitylation is dispensable for SG dynamics and that SGs co-localize primarily with free ubiquitin rather than polyubiquitylated proteins. Keywords: ubiquitin, stress granule, Nedd8, oxidative stress, sodium arsenite, protein homeostasis, centrosome, proteasome, protein aggregation, neurodegeneration
    Keywords Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2019-04-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Early transcriptional and epigenetic divergence of CD8+ T cells responding to acute versus chronic infection

    Lauren K. Quezada / Wenhao Jin / Yi Chia Liu / Eleanor S. Kim / Zhaoren He / Cynthia S. Indralingam / Tiffani Tysl / Lara Labarta-Bajo / Ellen J. Wehrens / Yeara Jo / Katelynn R. Kazane / Christopher Hattori / Elina I. Zuniga / Gene W. Yeo / John T. Chang

    PLoS Biology, Vol 21, Iss

    2023  Volume 1

    Abstract: During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the ... ...

    Abstract During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the context of chronic infections and cancer. Although it is evident that exhausted CD8+ T (TEX) cells are phenotypically and molecularly distinct from effector and memory CD8+ T cells, the factors regulating the earliest events in the differentiation process of TEX cells remain incompletely understood. Here, we performed single-cell RNA-sequencing and single-cell ATAC-sequencing of CD8+ T cells responding to LCMV-Armstrong (LCMV-Arm) or LCMV-Clone 13 (LCMV-Cl13), which result in acute or chronic infections, respectively. Compared to CD8+ T cells that had undergone their first division in response to LCMV-Arm (Div1ARM) cells, CD8+ T cells that had undergone their first division in response to LCMV-Cl13 (Div1CL13) expressed higher levels of genes encoding transcription factors previously associated with exhaustion, along with higher levels of Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex, which mediates epigenetic silencing. Modulation of Ezh2 resulted in altered expression of exhaustion-associated molecules by CD8+ T cells responding to LCMV-Cl13, though the specific cellular and infectious contexts, rather than simply the level of Ezh2 expression, likely determine the eventual outcome. Taken together, these findings suggest that the differentiation paths of CD8+ T cells responding to acute versus chronic infections may diverge earlier than previously appreciated. How early can the process of T cell exhaustion begin? This study shows that CD8+ T cells that have undergone their first cellular division in response to chronic versus acute infection may have already diverged along transcriptionally and epigenetically distinct differentiation paths.
    Keywords Biology (General) ; QH301-705.5
    Subject code 610
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: VPS13A and VPS13C Influence Lipid Droplet Abundance

    Shuliang Chen / Melissa A. Roberts / Chun-Yuan Chen / Sebastian Markmiller / Hong-Guang Wei / Gene W. Yeo / James G. Granneman / James A. Olzmann / Susan Ferro-Novick

    Contact, Vol

    2022  Volume 5

    Abstract: Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer ...

    Abstract Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson's disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) – lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate two lipid transfer proteins, VPS13A and VPS13C, in LD regulation.
    Keywords Biology (General) ; QH301-705.5 ; Biochemistry ; QD415-436
    Language English
    Publishing date 2022-09-01T00:00:00Z
    Publisher SAGE Publishing
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing

    Ryan J. Marina / Kristopher W. Brannan / Kevin D. Dong / Brian A. Yee / Gene W. Yeo

    Cell Reports, Vol 33, Iss 5, Pp 108350- (2020)

    2020  

    Abstract: Summary: Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA ... ...

    Abstract Summary: Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3′ modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3′ gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
    Keywords RNA editing ; Cas9 ; Cas13 ; ADAR2 ; CRISPR ; transcriptome engineering ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2020-11-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article: From Protein-RNA Predictions toward a Peptide-RNA Code

    Brannan, Kristopher W / Gene W. Yeo

    Molecular cell. 2016 Nov. 03, v. 64, no. 3

    2016  

    Abstract: The RNA field is undergoing a renaissance, with a deluge of proteins being identified to bind RNA. Two reports now introduce proteome-wide approaches that identify the peptides that are crosslinked to RNA (Castello et al., 2016; He et al., 2016). ...

    Abstract The RNA field is undergoing a renaissance, with a deluge of proteins being identified to bind RNA. Two reports now introduce proteome-wide approaches that identify the peptides that are crosslinked to RNA (Castello et al., 2016; He et al., 2016).
    Keywords crosslinking ; peptides ; prediction ; proteins ; RNA
    Language English
    Dates of publication 2016-1103
    Size p. 437-438.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1415236-8
    ISSN 1097-4164 ; 1097-2765
    ISSN (online) 1097-4164
    ISSN 1097-2765
    DOI 10.1016/j.molcel.2016.10.023
    Database NAL-Catalogue (AGRICOLA)

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