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1.
Cell ; 186(24): 5237-5253.e22, 2023 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-37944512

RESUMEN

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.


Asunto(s)
Cromosomas Artificiales de Levadura , Genoma Fúngico , Saccharomyces cerevisiae , Perfilación de la Expresión Génica , Proteómica , Saccharomyces cerevisiae/genética , Biología Sintética , ARN de Transferencia/genética , Cromosomas Artificiales de Levadura/genética
2.
Annu Rev Genet ; 57: 223-244, 2023 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-37562410

RESUMEN

Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades.


Asunto(s)
Sistemas CRISPR-Cas , Genoma , Sistemas CRISPR-Cas/genética , Genoma/genética , Pruebas Genéticas/métodos , Fenotipo
3.
Mol Syst Biol ; 19(3): e11254, 2023 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-36779527

RESUMEN

Microscopy and fluorescence-activated cell sorting (FACS) are two of the most important tools for single-cell phenotyping in basic and biomedical research. Microscopy provides high-resolution snapshots of cell morphology and the inner workings of cells, while FACS isolates thousands of cells per second using simple parameters, such as the intensity of fluorescent protein labels. Recent technologies are now combining both methods to enable the fast isolation of cells with microscopic phenotypes of interest, thereby bridging a long-standing gap in the life sciences. In this Commentary, we discuss the technical advancements made by image-enabled cell sorting and highlight novel experimental strategies in functional genomics and single-cell research.


Asunto(s)
Microscopía , Citometría de Flujo , Separación Celular
4.
Nat Methods ; 17(6): 629-635, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32483332

RESUMEN

The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer-target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma Humano , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Humanos
5.
Mol Syst Biol ; 17(4): e10232, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33904651

RESUMEN

Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.


Asunto(s)
Intestinos/inmunología , SARS-CoV-2/fisiología , Análisis de la Célula Individual , COVID-19/virología , Microbioma Gastrointestinal , Humanos , Hibridación Fluorescente in Situ , Organoides/metabolismo , Análisis de Secuencia de ARN
6.
PLoS Biol ; 17(3): e3000182, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30925180

RESUMEN

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.


Asunto(s)
Evolución Biológica , Escherichia coli/metabolismo , Humanos , Modelos Genéticos , Mutación/genética , Saccharomyces cerevisiae/metabolismo , Temperatura
7.
Trends Biochem Sci ; 39(9): 420-31, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25131816

RESUMEN

Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus.


Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Silenciador del Gen , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Argonautas/genética , Humanos , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética
8.
Proc Natl Acad Sci U S A ; 112(38): 11841-5, 2015 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-26351695

RESUMEN

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.


Asunto(s)
Proteínas Argonautas/aislamiento & purificación , Cromatografía de Afinidad/métodos , Complejos Multiproteicos/aislamiento & purificación , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Extractos Celulares , Precipitación Química , Drosophila melanogaster , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , MicroARNs/metabolismo , Datos de Secuencia Molecular , Péptidos/química
9.
Nucleic Acids Res ; 43(15): 7447-61, 2015 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-26170235

RESUMEN

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-ß pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.


Asunto(s)
Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ARN/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Autoantígenos/metabolismo , Línea Celular , Citoplasma/metabolismo , Silenciador del Gen , Humanos , Ratones
10.
EMBO J ; 30(20): 4309-22, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21857646

RESUMEN

Cancer stem cells or cancer initiating cells are believed to contribute to cancer recurrence after therapy. MicroRNAs (miRNAs) are short RNA molecules with fundamental roles in gene regulation. The role of miRNAs in cancer stem cells is only poorly understood. Here, we report miRNA expression profiles of glioblastoma stem cell-containing CD133(+) cell populations. We find that miR-9, miR-9(*) (referred to as miR-9/9(*)), miR-17 and miR-106b are highly abundant in CD133(+) cells. Furthermore, inhibition of miR-9/9(*) or miR-17 leads to reduced neurosphere formation and stimulates cell differentiation. Calmodulin-binding transcription activator 1 (CAMTA1) is a putative transcription factor, which induces the expression of the anti-proliferative cardiac hormone natriuretic peptide A (NPPA). We identify CAMTA1 as an miR-9/9(*) and miR-17 target. CAMTA1 expression leads to reduced neurosphere formation and tumour growth in nude mice, suggesting that CAMTA1 can function as tumour suppressor. Consistently, CAMTA1 and NPPA expression correlate with patient survival. Our findings could provide a basis for novel strategies of glioblastoma therapy.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Glioblastoma/metabolismo , MicroARNs/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Factor Natriurético Atrial/biosíntesis , Diferenciación Celular , Línea Celular Tumoral , Estudios de Cohortes , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/mortalidad , Glicoproteínas/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Transcriptoma
11.
Lab Chip ; 23(16): 3704-3715, 2023 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-37483015

RESUMEN

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Amilasas/metabolismo , Microfluídica , alfa-Amilasas/genética , alfa-Amilasas/metabolismo
12.
Nat Commun ; 14(1): 4312, 2023 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-37463913

RESUMEN

Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20's nuclear localization in RBM20-DCM patients.


Asunto(s)
Cardiomiopatía Dilatada , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Empalme del ARN/genética , Empalme Alternativo/genética , Mutación , Carioferinas/genética
13.
Science ; 375(6578): 315-320, 2022 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-35050652

RESUMEN

Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.


Asunto(s)
Citometría de Flujo , Imagen Óptica , Transporte Activo de Núcleo Celular , Animales , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Forma de la Célula , Técnicas Genéticas , Genoma , Genoma Humano , Humanos , Microscopía Fluorescente , Mitosis , FN-kappa B/metabolismo , Orgánulos/ultraestructura , Fenotipo , Factor de Transcripción ReIA/metabolismo
14.
Oncotarget ; 7(7): 7732-46, 2016 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-26741507

RESUMEN

Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-ß family of proteins. GDF-15 levels are increased in the blood and cerebrospinal fluid of glioblastoma patients. Using a TCGA database interrogation, we demonstrate that high GDF-15 expression levels are associated with poor survival of glioblastoma patients. To elucidate the role of GDF-15 in glioblastoma in detail, we confirmed that glioma cells express GDF-15 mRNA and protein in vitro. To allow for a detailed functional characterization, GDF-15 expression was silenced using RNA interference in LNT-229 and LN-308 glioma cells. Depletion of GDF-15 had no effect on cell viability. In contrast, GDF-15-deficient cells displayed reduced migration and invasion, in the absence of changes in Smad2 or Smad1/5/8 phosphorylation. Conversely, exogenous GDF-15 stimulated migration and invasiveness. Large-scale expression profiling revealed that GDF-15 gene silencing resulted in minor changes in the miRNA profile whereas several genes, including members of the plasminogen activator/inhibitor complex, were deregulated at the mRNA level. One of the newly identified genes induced by GDF-15 gene silencing was the serpin peptidase inhibitor, clade E nexin group 1 (serpine1) which is induced by TGF-ß and known to inhibit migration and invasiveness. However, serpine1 down-regulation alone did not mediate GDF-15-induced promotion of migration and invasiveness. Our findings highlight the complex contributions of GDF-15 to the invasive phenotype of glioma cells and suggest anti-GDF-15 approaches as a promising therapeutic strategy.


Asunto(s)
Neoplasias Encefálicas/patología , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Factor 15 de Diferenciación de Crecimiento/metabolismo , Apoptosis , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferación Celular , Perfilación de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Factor 15 de Diferenciación de Crecimiento/genética , Humanos , Técnicas para Inmunoenzimas , MicroARNs , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Células Tumorales Cultivadas
15.
J Cell Biol ; 207(4): 481-98, 2014 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-25404745

RESUMEN

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a "distribution box," transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.


Asunto(s)
ARN Ribosómico/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Escherichia coli/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Ribosómicas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Alineación de Secuencia
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