scholarly journals ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

2016 ◽  
Vol 212 (6) ◽  
pp. 647-659 ◽  
Author(s):  
Mayumi Isokane ◽  
Thomas Walter ◽  
Robert Mahen ◽  
Bianca Nijmeijer ◽  
Jean-Karim Hériché ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Genome Instability ◽  
Quantitative Imaging ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Genome Wide ◽  
A Genome

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment.

scholarly journals Integrative oncogene-dependency mapping identifies RIT1 vulnerabilities and synergies in lung cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Athea Vichas ◽  
Amanda K. Riley ◽  
Naomi T. Nkinsi ◽  
Shriya Kamlapurkar ◽  
Phoebe C. R. Parrish ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Genetic Data ◽  
Multiple Models ◽  
Ras Pathway ◽  
Functional Interactions ◽  
Genome Wide ◽  
A Genome ◽  
Mutant Cells

AbstractCRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals An integrative oncogene-dependency map identifies unique vulnerabilities of oncogenic EGFR, KRAS, and RIT1 in lung cancer

2020 ◽  
Author(s):  
Athea Vichas ◽  
Naomi T. Nkinsi ◽  
Amanda Riley ◽  
Phoebe C.R. Parrish ◽  
Fujiko Duke ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Spindle Assembly Checkpoint ◽  
Hippo Pathway ◽  
Spindle Assembly ◽  
Human Lung Cancer ◽  
Precision Oncology ◽  
Cancer Driver ◽  
Molecular Alterations ◽  
Pathway Gene ◽  
Genome Wide

ABSTRACTAdvances in precision oncology have transformed cancer therapy from broadly-applied cytotoxic therapy to personalized treatments based on each tumor’s unique molecular alterations. Here we investigate the oncogene-specific dependencies conferred by lung cancer driver variants of KRAS, EGFR, and RIT1. Integrative analysis of genome-wide CRISPR screens in isogenic cell lines identified shared and unique vulnerabilities of each oncogene. The non-identical landscape of dependencies underscores the importance of genotype-guided therapies to maximize tumor responses. Combining genetic screening data with small molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. This sensitivity may be related to a novel role of RIT1 in mitosis; we find that oncogenic RIT1M90I alters mitotic timing via weakening of the spindle assembly checkpoint. In addition, we uncovered a specific cooperation of mutant RIT1 with loss of Hippo pathway genes. In human lung cancer, RIT1 mutations and amplifications frequently co-occur with loss of Hippo pathway gene expression. These results provide the first genome-wide atlas of oncogenic RIT1-cooperating factors and genetic dependencies and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.

scholarly journals A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila

2010 ◽  
Vol 30 (11) ◽  
pp. 2837-2848 ◽  
Author(s):  
Vanessa Gobert ◽  
Dani Osman ◽  
Stéphanie Bras ◽  
Benoit Augé ◽  
Muriel Boube ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Rna Interference ◽  
Cell Fate ◽  
Gata Factor ◽  
Hematopoietic System ◽  
Crystal Cell ◽  
Genome Wide ◽  
A Genome

ABSTRACT Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory “CDK8 module,” composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.

scholarly journals Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

2016 ◽  
Vol 27 (11) ◽  
pp. 1753-1763 ◽  
Author(s):  
Hirohisa Masuda ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Synthetic Lethality ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Spindle Pole Bodies ◽  
Mitotic Spindle Pole

In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

scholarly journals A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells

2013 ◽  
Vol 23 (4) ◽  
pp. 581-591 ◽  
Author(s):  
K. Imberg-Kazdan ◽  
S. Ha ◽  
A. Greenfield ◽  
C. S. Poultney ◽  
R. Bonneau ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Rna Interference ◽  
Cancer Cells ◽  
Receptor Function ◽  
Prostate Cancer Cells ◽  
Genome Wide ◽  
A Genome ◽  
Androgen Receptor Function

scholarly journals A Genome-wide RNA Interference Screen Reveals that Variant Histones Are Necessary for Replication-Dependent Histone Pre-mRNA Processing

2007 ◽  
Vol 28 (4) ◽  
pp. 692-699 ◽  
Author(s):  
Eric J. Wagner ◽  
Brandon D. Burch ◽  
Ashley C. Godfrey ◽  
Harmony R. Salzler ◽  
Robert J. Duronio ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mrna Processing ◽  
Genome Wide ◽  
A Genome

scholarly journals Regulation of Mitotic Spindle Asymmetry by SUMO and the Spindle-Assembly Checkpoint in Yeast

2008 ◽  
Vol 18 (16) ◽  
pp. 1249-1255 ◽  
Author(s):  
Christian Leisner ◽  
Daniel Kammerer ◽  
Annina Denoth ◽  
Mirjam Britschi ◽  
Yves Barral ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly

scholarly journals A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection

2016 ◽  
Vol 90 (16) ◽  
pp. 7084-7097 ◽  
Author(s):  
Brooke Harmon ◽  
Sara W. Bird ◽  
Benjamin R. Schudel ◽  
Anson V. Hatch ◽  
Amy Rasley ◽  
...  
Keyword(s):  
Rna Interference ◽  
Wnt Signaling ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Valley Fever ◽  
Rnai Screening ◽  
Genome Wide ◽  
A Genome

ABSTRACTRift Valley fever virus (RVFV) is an arbovirus within theBunyaviridaefamily capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics.IMPORTANCERVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

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