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Anna M McGeachy

Bio: Anna M McGeachy is an academic researcher from University of California, Berkeley. The author has contributed to research in topics: Translation (biology) & Ribosome profiling. The author has an hindex of 5, co-authored 9 publications receiving 1378 citations. Previous affiliations of Anna M McGeachy include Thermo Fisher Scientific & Carnegie Institution for Science.

Papers
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Journal ArticleDOI
TL;DR: An adaptation that reveals the sites of translation initiation by pretreating cells with harringtonine to immobilize initiating ribosomes is described, paving the way for genome-wide, quantitative analysis of in vivo translation by deep sequencing.
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.

1,080 citations

Journal ArticleDOI
26 Feb 2015-eLife
TL;DR: By genome-wide in vivo ribosome profiling, ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells.
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.

432 citations

Reference EntryDOI
TL;DR: An adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes is described, paving the way for genome-wide, quantitative analysis of in vivo translation by deep sequencing.
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.

67 citations

Journal ArticleDOI
TL;DR: An accessible, robust continuous-culture turbidostat system that greatly facilitates the generation and phenotypic analysis of highly complex libraries in yeast and bacteria and performs a comprehensive mutational analysis of the essential gene RPL28 in budding yeast is presented.
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.

26 citations


Cited by
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Journal ArticleDOI
TL;DR: The function of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis, with a particular emphasis on mammalian development are described.
Abstract: Genomes of multicellular organisms are characterized by the pervasive expression of different types of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs) belong to a novel heterogeneous class of ncRNAs that includes thousands of different species. lncRNAs have crucial roles in gene expression control during both developmental and differentiation processes, and the number of lncRNA species increases in genomes of developmentally complex organisms, which highlights the importance of RNA-based levels of control in the evolution of multicellular organisms. In this Review, we describe the function of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis, with a particular emphasis on mammalian development.

2,464 citations

Journal ArticleDOI
TL;DR: Current understanding of the ISR signaling is reviewed and how it regulates cell fate under diverse types of stress is reviewed.
Abstract: In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro‐survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.

1,480 citations

Journal ArticleDOI
27 Feb 2015-Science
TL;DR: It is shown that N6-methyladenosine (m6A), a messenger RNA (mRNA) modification present on transcripts of pluripotency factors, drives this transition from the pluripotent to the differentiated state.
Abstract: Naive and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naive pluripotency. Mettl3 knockout preimplantation epiblasts and naive embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naive state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naive pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naive and primed pluripotency in an opposing manner.

1,181 citations

Journal ArticleDOI
24 Apr 2014-Cell
TL;DR: This work presents a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates, and reveals how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from quantitative analyses of protein synthesis.

1,115 citations

Journal ArticleDOI
TL;DR: The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of annotated genomic, transcript and protein sequence records derived from data in public sequence archives and from computation, curation and collaboration.
Abstract: The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of annotated genomic, transcript and protein sequence records derived from data in public sequence archives and from computation, curation and collaboration (http://wwwncbinlmnihgov/refseq/) We report here on growth of the mammalian and human subsets, changes to NCBI’s eukaryotic annotation pipeline and modifications affecting transcript and protein records Recent changes to NCBI’s eukaryotic genome annotation pipeline provide higher throughput, and the addition of RNAseq data to the pipeline results in a significant expansion of the number of transcripts and novel exons annotated on mammalian RefSeq genomes Recent annotation changes include reporting supporting evidence for transcript records, modification of exon feature annotation and the addition of a structured report of gene and sequence attributes of biological interest We also describe a revised protein annotation policy for alternatively spliced transcripts with more divergent predicted proteins and we summarize the current status of the RefSeqGene project

949 citations