RESUMEN
RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.
Asunto(s)
Biosíntesis de Proteínas , Virus ARN/fisiología , Replicación Viral/fisiología , Línea Celular Tumoral , Supervivencia Celular , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferones/metabolismo , Transporte de ARN , ARN Viral/genética , Reproducibilidad de los Resultados , Imagen Individual de Molécula , Factores de TiempoRESUMEN
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Asunto(s)
Colorantes Fluorescentes/química , ARN Mensajero/metabolismo , Imagen Individual de Molécula/métodos , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Línea Celular Tumoral , Genes Reporteros , Células HEK293 , Humanos , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , Ribosomas/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.
Asunto(s)
Imagen Óptica/métodos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Fluorescencia , Genes Reporteros , Técnicas Genéticas , Proteínas Fluorescentes Verdes/análisis , Humanos , Proteínas Luminiscentes/análisis , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , Ribosomas/metabolismo , Proteína Fluorescente RojaRESUMEN
Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.
Asunto(s)
Imagen Molecular/métodos , Imagen Óptica/métodos , Multimerización de Proteína , Proteínas/química , Animales , Sistemas CRISPR-Cas , Técnicas Genéticas , Humanos , Anticuerpos de Cadena Única/químicaRESUMEN
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Neoplasias/patología , Preparaciones Farmacéuticas/metabolismo , Sulfonas/farmacología , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Contaminación de Medicamentos , Glicina/farmacología , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Preparaciones Farmacéuticas/química , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
Asunto(s)
Codón sin Sentido/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , Codón sin Sentido/química , Exones/genética , Humanos , Intrones/genética , Mutación/genética , Estabilidad del ARN/genética , ARN Mensajero/química , Imagen Individual de MoléculaRESUMEN
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
Asunto(s)
Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Pruebas Genéticas/métodos , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Sulfonas/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/genética , Antineoplásicos/química , Sistemas CRISPR-Cas , Colchicina/farmacología , Resistencia a Antineoplásicos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicina/química , Glicina/farmacología , Células HeLa , Humanos , Células K562 , Cinesinas/genética , Cinesinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vinblastina/farmacologíaRESUMEN
Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA-protein complexes that enable live imaging of mRNA in living cells.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , ARN Mensajero/análisis , Aptámeros de Nucleótidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismoRESUMEN
The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors' velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of "identical" motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single-molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub-ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte-Carlo multi-motor simulations.
Asunto(s)
Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Simulación de Dinámica Molecular , Animales , Línea Celular Tumoral , Drosophila , Proteínas de Drosophila/química , Humanos , Cinesinas/química , Movimiento (Física) , Dominios ProteicosRESUMEN
Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.
Asunto(s)
Aurora Quinasa A/metabolismo , Cinesinas/metabolismo , Microtúbulos , Huso Acromático , Estudio de Asociación del Genoma Completo , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis , ARN Interferente Pequeño/genética , Huso Acromático/metabolismoRESUMEN
The microtubule motor protein kinesin-5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti-cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5-independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)-associated dynein that drives centrosome separation in prophase. This NE-dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5-dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin-5 inhibitors.
Asunto(s)
Centrosoma/fisiología , Dineínas/metabolismo , Cinesinas/metabolismo , Mitosis/fisiología , Membrana Nuclear/fisiología , Profase/fisiología , Huso Acromático/fisiología , Western Blotting , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Ensayo de Unidades Formadoras de Colonias , Dineínas/genética , Citometría de Flujo , Células HeLa , Humanos , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , ARN Interferente Pequeño/genéticaRESUMEN
Respiratory syncytial virus (RSV) is the most prevalent cause of acute lower respiratory infection in young children. Currently, the first RSV vaccines are approved by the FDA. Recently, N6-methyladenosine (m6A) RNA methylation has been implicated in the regulation of the viral life cycle and replication of many viruses, including RSV. m6A methylation of RSV RNA has been demonstrated to promote replication and prevent anti-viral immune responses by the host. Whether m6A is also involved in viral entry and whether m6A can also affect RSV infection via different mechanisms than methylation of viral RNA is poorly understood. Here, we identify m6A reader YTH domain-containing protein 1 (YTHDC1) as a novel negative regulator of RSV infection. We demonstrate that YTHDC1 abrogates RSV infection by reducing the expression of RSV entry receptor CX3C motif chemokine receptor 1 (CX3CR1) on the cell surface of lung epithelial cells. Altogether, these data reveal a novel role for m6A methylation and YTHDC1 in the viral entry of RSV. These findings may contribute to the development of novel treatment options to control RSV infection.
Asunto(s)
Receptor 1 de Quimiocinas CX3C , Regulación hacia Abajo , Factores de Empalme de ARN , Infecciones por Virus Sincitial Respiratorio , Humanos , Células A549 , Adenosina/análogos & derivados , Adenosina/metabolismo , Línea Celular , Receptor 1 de Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Células Epiteliales/virología , Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno , Metilación , Proteínas del Tejido Nervioso , Infecciones por Virus Sincitial Respiratorio/virología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitial Respiratorio Humano/fisiología , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , ARN Viral/genética , ARN Viral/metabolismo , Internalización del Virus , Replicación ViralRESUMEN
BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Mitosis/fisiología , Poro Nuclear/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Núcleo Celular/ultraestructura , Complejo Dinactina , Humanos , Cinesinas/genética , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.
Asunto(s)
Microscopía , Nanotecnología , Nanotecnología/métodos , Imagen Individual de Molécula , Espectrometría de Fluorescencia/métodosRESUMEN
Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation.
Asunto(s)
Virosis , Replicación Viral , Humanos , Transducción de Señal , Virus de la Encefalomiocarditis/fisiología , AntiviralesRESUMEN
Efficient spindle assembly involves the generation of spatial cues around chromosomes that locally stabilize microtubule (MT) plus-ends. In addition to the small GTPase Ran, there is evidence that Aurora B kinase might also generate a spatial cue around chromosomes but direct proof for this is still lacking. Here, we find that the Aurora B substrate MCAK localizes to MT plus-ends throughout the mitotic spindle, but its accumulation is strongly reduced on MT plus-ends near chromatin, suggesting that a signal emanating from chromosomes negatively regulates MCAK plus-end binding. Indeed, we show that Aurora B is the kinase responsible for producing this chromosome-derived signal. These results are the first to visualize spatially restricted Aurora B kinase activity around chromosomes on an endogenous substrate and explain how Aurora B could spatially control the dynamics of non-kinetochore MTs during spindle assembly.
Asunto(s)
Células/citología , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Aurora Quinasa B , Aurora Quinasas , Línea Celular , Células/enzimología , Células/metabolismo , Humanos , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Huso Acromático/enzimología , Huso Acromático/genéticaRESUMEN
Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , División Celular , Centrosoma/metabolismo , Dineínas/metabolismo , Cinesinas/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Línea Celular , Humanos , Modelos BiológicosRESUMEN
Accurate control of the cell cycle is critical for development and tissue homeostasis, and requires precisely timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle in human cells. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and we identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.
Asunto(s)
Ciclo Celular/genética , Ciclo Celular/fisiología , Estabilidad del ARN/fisiología , Línea Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.
Asunto(s)
Clatrina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Línea Celular Tumoral , Centrómero/metabolismo , Segregación Cromosómica/fisiología , Cromosomas Humanos/genética , Cromosomas Humanos/fisiología , Cromosomas Humanos/ultraestructura , Clatrina/genética , Clatrina/fisiología , Células HeLa , Humanos , Cinetocoros/fisiología , Microtúbulos/ultraestructura , Mitosis , ARN Interferente Pequeño , Huso Acromático/genética , Tubulina (Proteína)/metabolismoRESUMEN
RNA-based therapeutics are highly promising for the treatment of numerous diseases, by their ability to tackle the genetic origin in multiple possible ways. RNA molecules are, however, incapable of crossing cell membranes, hence a safe and efficient delivery vehicle is pivotal. Extracellular vesicles (EVs) are endogenously derived nano-sized particles and possess several characteristics which make them excellent candidates as therapeutic RNA delivery agent. This includes the inherent capability to functionally transfer RNAs in a selective manner and an enhanced safety profile compared to synthetic particles. Nonetheless, the fundamental mechanisms underlying this selective inter- and intracellular trafficking and functional transfer of RNAs by EVs are poorly understood. Improving our understanding of these systems is a key element of working towards an EV-based or EV-mimicking system for the functional delivery of therapeutic RNA. In this review, state-of-the-art approaches to detect and visualize RNA in situ and in live cells are discussed, as well as strategies to assess functional RNA transfer, highlighting their potential in studying EV-RNA trafficking mechanisms.