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1.
Cell ; 187(6): 1490-1507.e21, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38452761

RESUMO

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucarióticas/metabolismo , Redes Neurais de Computação , Proteoma/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Nature ; 533(7604): 499-503, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27225121

RESUMO

Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored. Here we monitor mitochondrial and nuclear gene expression in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. We show that nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, mitochondrial and cytosolic translation are rapidly, dynamically and synchronously regulated. Furthermore, cytosolic translation processes control mitochondrial translation unidirectionally. Thus, the nuclear genome coordinates mitochondrial and cytosolic translation to orchestrate the timely synthesis of OXPHOS complexes, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for studies of global gene regulation in mitochondria.


Assuntos
Núcleo Celular/genética , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/biossíntese , Fosforilação Oxidativa , Biossíntese de Proteínas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Genes Mitocondriais/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Biogênese de Organelas , Proteoma/biossíntese , Proteoma/genética , RNA Fúngico/análise , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
Mol Cell ; 48(4): 509-20, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23084833

RESUMO

Emerging evidence suggests that Argonaute (Ago)/Piwi proteins have diverse functions in the nucleus and cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound small RNAs (sRNAs) are 3' tRNA fragments that contain modified bases and thus are attenuated for base pairing to targets. We show that Twi12 assembles an unexpected complex with the nuclear exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2, as well as to stimulate its exonuclease activity. Twi12 function depends on sRNA binding, which is required for its nuclear import. Depletion of Twi12 or Xrn2 induces a cellular ribosomal RNA processing defect known to result from limiting Xrn2 activity in other organisms. Our findings suggest a role for an Ago/Piwi protein and 3' tRNA fragments in nuclear RNA metabolism.


Assuntos
Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Tetrahymena/genética , Proteínas Argonautas/genética , Núcleo Celular/enzimologia , Núcleo Celular/genética , RNA de Transferência/genética , Tetrahymena/citologia , Tetrahymena/metabolismo
4.
Genes Dev ; 24(24): 2742-7, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21106669

RESUMO

Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18- to 22-nucleotide tRNA 3' fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA fragment small RNAs.


Assuntos
Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Tetrahymena/genética , Sequência de Bases , Evolução Biológica , Proteínas de Protozoários/fisiologia , Tetrahymena/crescimento & desenvolvimento
5.
Genes Dev ; 23(17): 2016-32, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19656801

RESUMO

PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23- to 24-nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether, Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length, while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Animais , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/química , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Ligação Proteica , RNA de Protozoário/química , Análise de Sequência de RNA , Proteína 1 Relacionada a Twist/metabolismo
6.
BMC Genomics ; 9: 562, 2008 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-19036158

RESUMO

BACKGROUND: Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. RESULTS: We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. CONCLUSION: We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.


Assuntos
Genoma de Protozoário , Tetrahymena thermophila/genética , Animais , Etiquetas de Sequências Expressas , Genes de Protozoários , Macronúcleo , Micronúcleo Germinativo , Hibridização de Ácido Nucleico
7.
Curr Protoc Mol Biol ; 119: 4.28.1-4.28.25, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28678443

RESUMO

Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome. As information about RNA abundance is required to resolve translational from RNA effects, we also present an RNA sequencing approach that can be performed in parallel. Accurately capturing the biologically relevant distribution of mitoribosome positions depends on several critical parameters that are discussed. Application of mitoribosome profiling can reveal mechanisms of mitochondrial translational control that were not previously possible to uncover. © 2017 by John Wiley & Sons, Inc.


Assuntos
Perfilação da Expressão Gênica/métodos , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Northern Blotting , Imunoprecipitação , Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA
8.
Methods Cell Biol ; 109: 347-55, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22444151

RESUMO

Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit. Common biochemical methods for detecting protein, RNA, and DNA in Tetrahymena thermophila are also discussed.


Assuntos
Marcadores de Afinidade/química , Mapeamento de Epitopos/métodos , Epitopos/química , Técnicas Imunológicas/métodos , Proteínas de Protozoários/isolamento & purificação , Tetrahymena thermophila/química , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Loci Gênicos , Macronúcleo/química , Macronúcleo/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Organismos Geneticamente Modificados/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , Tetrahymena thermophila/genética , Transgenes
9.
G3 (Bethesda) ; 1(6): 515-22, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22384362

RESUMO

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3' exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.

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