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  1. Article ; Online: Surveying the global landscape of post-transcriptional regulators.

    Reynaud, Kendra / McGeachy, Anna M / Noble, David / Meacham, Zuriah A / Ingolia, Nicholas T

    Nature structural & molecular biology

    2023  Volume 30, Issue 6, Page(s) 740–752

    Abstract: Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome ...

    Abstract Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.
    MeSH term(s) Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/analysis ; Saccharomyces cerevisiae Proteins/metabolism ; Proteome ; RNA, Messenger/chemistry ; RNA, Messenger/metabolism ; RNA Processing, Post-Transcriptional ; Gene Expression Regulation ; RNA Stability ; RNA-Binding Proteins/analysis ; RNA-Binding Proteins/metabolism
    Chemical Substances Saccharomyces cerevisiae Proteins ; Proteome ; RNA, Messenger ; RNA-Binding Proteins
    Language English
    Publishing date 2023-05-25
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2126708-X
    ISSN 1545-9985 ; 1545-9993
    ISSN (online) 1545-9985
    ISSN 1545-9993
    DOI 10.1038/s41594-023-00999-5
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 4101: Show issues Location:
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    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)
    Zs.MG 56: Show issues

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  2. Article ; Online: An Accessible Continuous-Culture Turbidostat for Pooled Analysis of Complex Libraries.

    McGeachy, Anna M / Meacham, Zuriah A / Ingolia, Nicholas T

    ACS synthetic biology

    2019  Volume 8, Issue 4, Page(s) 844–856

    Abstract: We present an accessible, robust continuous-culture turbidostat system that greatly facilitates the generation and phenotypic analysis of highly complex libraries in yeast and bacteria. Our system has many applications in genomics and systems biology; ... ...

    Abstract We present an accessible, robust continuous-culture turbidostat system that greatly facilitates the generation and phenotypic analysis of highly complex libraries in yeast and bacteria. Our system has many applications in genomics and systems biology; here, we demonstrate three of these uses. We first measure how the growth rate of budding yeast responds to limiting nitrogen at steady state and in a dynamically varying environment. We also demonstrate the direct selection of a diverse, genome-scale protein fusion library in liquid culture. Finally, we perform a comprehensive mutational analysis of the essential gene RPL28 in budding yeast, mapping sequence constraints on its wild-type function and delineating the binding site of the drug cycloheximide through resistance mutations. Our system can be constructed and operated with no specialized skills or equipment and applied to study genome-wide mutant pools and diverse libraries of sequence variants under well-defined growth conditions.
    MeSH term(s) Bacteria/genetics ; Binding Sites/genetics ; Cell Culture Techniques/methods ; Genes, Essential/genetics ; Genome/genetics ; Genomics/methods ; Mutation/genetics ; Saccharomyces cerevisiae/genetics
    Language English
    Publishing date 2019-04-02
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 2161-5063
    ISSN (online) 2161-5063
    DOI 10.1021/acssynbio.8b00529
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  3. Article ; Online: Starting too soon: upstream reading frames repress downstream translation.

    McGeachy, Anna M / Ingolia, Nicholas T

    The EMBO journal

    2016  Volume 35, Issue 7, Page(s) 699–700

    Abstract: Upstream open reading frames (uORFs) are known to regulate a few specific transcripts, and recent computational and experimental studies have suggested candidate uORF regulation across the genome. In this issue, Johnstone et al (2016) use ribosome ... ...

    Abstract Upstream open reading frames (uORFs) are known to regulate a few specific transcripts, and recent computational and experimental studies have suggested candidate uORF regulation across the genome. In this issue, Johnstone et al (2016) use ribosome profiling to identify translated uORFs and measure their effects on downstream translation. Furthermore, they show that regulatory uORFs are conserved across species and subject to selective constraint. Recognizing the potential of uORFs in regulating translation expands our understanding of the dynamic regulation of gene expression.
    MeSH term(s) Animals ; Open Reading Frames ; Protein Biosynthesis ; Repressor Proteins/metabolism ; Vertebrates/genetics
    Chemical Substances Repressor Proteins
    Language English
    Publishing date 2016-04-01
    Publishing country England
    Document type Comment ; Journal Article
    ZDB-ID 586044-1
    ISSN 1460-2075 ; 0261-4189
    ISSN (online) 1460-2075
    ISSN 0261-4189
    DOI 10.15252/embj.201693946
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    Zs.A 1808: Show issues Location:
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    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)
    Zs.MG 100: Show issues

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  4. Article ; Online: The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.

    Sidrauski, Carmela / McGeachy, Anna M / Ingolia, Nicholas T / Walter, Peter

    eLife

    2015  Volume 4

    Abstract: Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we ... ...

    Abstract Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.
    MeSH term(s) Acetamides/pharmacology ; Animals ; Cyclohexylamines/pharmacology ; Cytoplasmic Granules/drug effects ; Eukaryotic Initiation Factor-2/drug effects ; Eukaryotic Initiation Factor-2/metabolism ; Phosphorylation ; Protein Biosynthesis/drug effects ; RNA, Messenger/metabolism ; Ribosomes/metabolism ; Stress, Physiological
    Chemical Substances 2-(4-chlorophenoxy)-N-(4-(2-(4-chlorophenoxy)acetamido)cyclohexyl)acetamide ; Acetamides ; Cyclohexylamines ; Eukaryotic Initiation Factor-2 ; RNA, Messenger
    Language English
    Publishing date 2015-02-26
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.05033
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  5. Article ; Online: Genome-wide annotation and quantitation of translation by ribosome profiling.

    Ingolia, Nicholas T / Brar, Gloria A / Rouskin, Silvia / McGeachy, Anna M / Weissman, Jonathan S

    Current protocols in molecular biology

    2013  Volume Chapter 4, Page(s) 4.18.1–4.18.19

    Abstract: Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. A protocol for genome-wide, quantitative analysis of in vivo translation ... ...

    Abstract Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. A protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing is presented here. This ribosome-profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes is described. The protocol described requires 5 to 7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis requires an additional 4 to 5 days.
    MeSH term(s) Computational Biology/methods ; Gene Expression Profiling/methods ; High-Throughput Nucleotide Sequencing/methods ; Protein Biosynthesis ; Ribosomes/metabolism ; Time Factors
    Language English
    Publishing date 2013-07-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 1934-3647
    ISSN (online) 1934-3647
    DOI 10.1002/0471142727.mb0418s103
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  6. Article ; Online: The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly

    Carmela Sidrauski / Anna M McGeachy / Nicholas T Ingolia / Peter Walter

    eLife, Vol

    2015  Volume 4

    Abstract: Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we ... ...

    Abstract Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.
    Keywords integrated stress response ; eIF2 ; unfolded protein response ; ISRIB ; protein synthesis ; ribosome profiling ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2015-02-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.

    Ingolia, Nicholas T / Brar, Gloria A / Rouskin, Silvia / McGeachy, Anna M / Weissman, Jonathan S

    Nature protocols

    2012  Volume 7, Issue 8, Page(s) 1534–1550

    Abstract: Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo ... ...

    Abstract Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. In addition, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.
    MeSH term(s) Animals ; Base Sequence ; Gene Library ; Harringtonines/pharmacology ; Humans ; Molecular Sequence Data ; Peptide Chain Initiation, Translational ; Protein Biosynthesis/genetics ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Ribosomal ; Ribonucleases/metabolism ; Ribosomes/drug effects ; Ribosomes/genetics ; Ribosomes/metabolism ; Saccharomyces cerevisiae/cytology ; Sequence Analysis, RNA/methods ; Transcriptome
    Chemical Substances Harringtonines ; RNA, Messenger ; RNA, Ribosomal ; harringtonin (088662H40F) ; Ribonucleases (EC 3.1.-)
    Language English
    Publishing date 2012-07-26
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/nprot.2012.086
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  8. Article ; Online: Oral epithelial cells orchestrate innate type 17 responses to

    Verma, Akash H / Richardson, Jonathan P / Zhou, Chunsheng / Coleman, Bianca M / Moyes, David L / Ho, Jemima / Huppler, Anna R / Ramani, Kritika / McGeachy, Mandy J / Mufazalov, Ilgiz A / Waisman, Ari / Kane, Lawrence P / Biswas, Partha S / Hube, Bernhard / Naglik, Julian R / Gaffen, Sarah L

    Science immunology

    2017  Volume 2, Issue 17

    Abstract: ... Candida ... ...

    Abstract Candida albicans
    MeSH term(s) Adaptive Immunity/immunology ; Animals ; Candida albicans/immunology ; Candida albicans/metabolism ; Candida albicans/physiology ; Candidiasis/immunology ; Candidiasis/microbiology ; Cytokines/immunology ; Cytokines/metabolism ; Epithelial Cells/immunology ; Epithelial Cells/microbiology ; Female ; Fungal Proteins/genetics ; Fungal Proteins/immunology ; Fungal Proteins/metabolism ; Humans ; Hyphae/immunology ; Hyphae/metabolism ; Hyphae/physiology ; Immunity, Innate/immunology ; Interleukin-17/genetics ; Interleukin-17/immunology ; Interleukin-17/metabolism ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Transgenic ; Mouth Mucosa/immunology ; Mouth Mucosa/microbiology ; Virulence Factors/genetics ; Virulence Factors/immunology ; Virulence Factors/metabolism
    Chemical Substances Cytokines ; ECE1 protein, Candida albicans ; Fungal Proteins ; Interleukin-17 ; Virulence Factors
    Language English
    Publishing date 2017-11-01
    Publishing country United States
    Document type Journal Article
    ISSN 2470-9468
    ISSN (online) 2470-9468
    DOI 10.1126/sciimmunol.aam8834
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