Validating Eif4a1 by CRISPR-Cas9 Knockout As a Potential Target For

Total Page：16

File Type：pdf, Size：1020Kb

Validating eIF4A1 by CRISPR-Cas9 knockout as a potential target for cancer therapeutics Anna Bakewell1, Zaynab Neetoo-Isseljee1, Craig Southern1, Barbara Saxty1, Debra Taylor1 1LifeArc, Accelerator Building, SBC Campus, Stevenage, SG1 2FX, UK Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent DEAD/H box RNA helicase involved in translation initiation. Overexpression of eIF4A is observed in multiple cancer cell lines and is thought to accommodate the upregulation of translation of pro-oncogenic mRNA, thus contributing to oncogenesis. Studies have demonstrated that suppressing eIF4A1, one of the two isoforms of eIF4A that configure part of the eIF4F complex, results in reduced growth and survival of cancer cells, which is not observed when eIF4A2 is knocked down. The activity of eIF4A1 can be enhanced by pro-oncogenic transcription factors increasing translation of eIF4A1-dependent mRNAs. Many of these mRNAs encode pro-proliferative proteins involved in the cell cycle, cell survival and angiogenesis and contain highly structured 5’-UTRs containing G-quadruplexes. eIF4A unwinds these secondary structures enabling the 43S pre-initiation complex to bind to the mRNA and begin moving along it until it recognises the AUG start codon, at which point irreversible GTP hydrolysis of eIF42 prevents further unwinding and association of the 60S ribosomal subunit occurs allowing chain elongation to commence. In order to provide target validation of eIF4A1 we utilised emerging CRISPR-Cas9 gene editing technology to generate knockouts of two of the isoforms in isolation and simultaneously and developed a transient lethality assay. The gene knockouts were generated in breast cancer cell lines, MDA-MB-231 and MCF-7, using ribonucleoprotein complexes comprising Cas9 nuclease and synthetic multi-guide RNA specific to eIF4A1 and eIF4A2 genes. The results from the phenotypic assay demonstrated the effects of knocking out related genes simultaneously. Utilising CRISPR-Cas9 gene editing technology to knockout genes and develop transient lethality assays has proved to be advantageous over RNAi knockdown which we compared in- house to further validate this target. .

Copy Link

Recommended publications

A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
[Show full text]
Early Alterations of RNA Metabolism and Splicing from Adult Corticospinal Neurons In
bioRxiv preprint doi: https://doi.org/10.1101/667733; this version posted June 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Early alterations of RNA metabolism and splicing from adult corticospinal neurons in 2 an ALS mouse model 3 4 Christine Marques1,2, Mathieu Fischer1,3, Céline Keime4, Thibaut Burg1, Aurore Brunet1, 5 Jelena Scekic-Zahirovic1 & Caroline Rouaux1* 6 7 8 9 1Inserm UMR_S 1118, Mécanismes centraux et périphériques de la neurodégénérescence, 10 Faculté de Médecine, Université de Strasbourg, Strasbourg, France. 11 2Current address: Department of Neurobiology, Harvard Medical School, Boston, MA, USA; 12 Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. 13 3Current address: Department of Paediatrics, John Radcliffe Hospital, University of Oxford, 14 Oxford, UK. 15 4Inserm UMR_S 1258, CRNS UMR_S 7104, Université de Strasbourg, IGBMC, Strasbourg, 16 France. 17 18 *Correspondence should be addressed to: C.R. ([email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/667733; this version posted June 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease clinically defined as the combined degeneration of corticospinal and corticobulbar neurons (CSN), and bulbar and spinal motor neurons (MN). A growing body of evidence points to the motor cortex, where CSN are located, as the potential initiation site of ALS. However, little is known about the spatiotemporal dynamics of CSN degeneration and the molecular pathways involved.
[Show full text]
Supplementary Material Contents
Supplementary Material Contents Immune modulating proteins identified from exosomal samples.....................................................................2 Figure S1: Overlap between exosomal and soluble proteomes.................................................................................... 4 Bacterial strains:..............................................................................................................................................4 Figure S2: Variability between subjects of effects of exosomes on BL21-lux growth.................................................... 5 Figure S3: Early effects of exosomes on growth of BL21 E. coli .................................................................................... 5 Figure S4: Exosomal Lysis............................................................................................................................................ 6 Figure S5: Effect of pH on exosomal action.................................................................................................................. 7 Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant ....... 8 Effective exosomal concentration....................................................................................................................8 Figure S7: Sample constitution for luminometry experiments..................................................................................... 8 Figure S8: Determining effective concentration .........................................................................................................
[Show full text]
Relevance of Translation Initiation in Diffuse Glioma Biology and Its
cells Review Relevance of Translation Initiation in Diﬀuse Glioma Biology and its Therapeutic Potential Digregorio Marina 1, Lombard Arnaud 1,2, Lumapat Paul Noel 1, Scholtes Felix 1,2, Rogister Bernard 1,3 and Coppieters Natacha 1,* 1 Laboratory of Nervous System Disorders and Therapy, GIGA-Neurosciences Research Centre, University of Liège, 4000 Liège, Belgium; [email protected] (D.M.); [email protected] (L.A.); [email protected] (L.P.N.); [email protected] (S.F.); [email protected] (R.B.) 2 Department of Neurosurgery, CHU of Liège, 4000 Liège, Belgium 3 Department of Neurology, CHU of Liège, 4000 Liège, Belgium * Correspondence: [email protected] Received: 18 October 2019; Accepted: 26 November 2019; Published: 29 November 2019 Abstract: Cancer cells are continually exposed to environmental stressors forcing them to adapt their protein production to survive. The translational machinery can be recruited by malignant cells to synthesize proteins required to promote their survival, even in times of high physiological and pathological stress. This phenomenon has been described in several cancers including in gliomas. Abnormal regulation of translation has encouraged the development of new therapeutics targeting the protein synthesis pathway. This approach could be meaningful for glioma given the fact that the median survival following diagnosis of the highest grade of glioma remains short despite current therapy. The identiﬁcation of new targets for the development of novel therapeutics is therefore needed in order to improve this devastating overall survival rate. This review discusses current literature on translation in gliomas with a focus on the initiation step covering both the cap-dependent and cap-independent modes of initiation.
[Show full text]
Loss of Eif4e Phosphorylation Engenders Depression-Like Behaviors Via Selective Mrna Translation
This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Neurobiology of Disease Loss of eIF4E phosphorylation engenders depression-like behaviors via selective mRNA translation Inês Silva Amorim1,2, Sonal Kedia1,2, Stella Kouloulia1,2, Konstanze Simbriger1,2, Ilse Gantois3, Seyed Mehdi Jafarnejad3, Yupeng Li1,2, Agniete Kampaite1,2, Tine Pooters1, Nicola Romanò1 and Christos G. Gkogkas1,2,4 1Centre for Discovery Brain Sciences, EH8 9XD, Edinburgh, Scotland, UK 2The Patrick Wild Centre, EH8 9XD, Edinburgh, Scotland, UK 3Goodman Cancer Research Centre and Biochemistry Department, McGill University, H3A 1A3, Montréal, QC, Canada 4Simons Initiative for the Developing Brain, EH8 9XD, Edinburgh, Scotland, UK DOI: 10.1523/JNEUROSCI.2673-17.2018 Received: 16 September 2017 Revised: 3 December 2017 Accepted: 8 January 2018 Published: 24 January 2018 Author contributions: I.S.A., S. Kedia, S. Kouloulia, S.M.J., N.R., and C.G.G. designed research; I.S.A., S. Kedia, S. Kouloulia, K.S., I.G., S.M.J., Y.L., A.K., N.R., and C.G.G. performed research; I.S.A., S. Kedia, S. Kouloulia, K.S., I.G., S.M.J., A.K., T.P., and C.G.G. analyzed data; N.R. and C.G.G. contributed unpublished reagents/analytic tools; C.G.G. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by grants to CGG: Sir Henry Dale Fellowship from the Wellcome Trust and Royal Society (107687/Z/15/Z), a NARSAD Young Investigator grant from the Brain & Behavior Research Foundation, the RS Macdonald Charitable Trust and the Patrick Wild Centre.
[Show full text]
Inhibition of the MID1 Protein Complex
Matthes et al. Cell Death Discovery (2018) 4:4 DOI 10.1038/s41420-017-0003-8 Cell Death Discovery ARTICLE Open Access Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis Frank Matthes1,MoritzM.Hettich1, Judith Schilling1, Diana Flores-Dominguez1, Nelli Blank1, Thomas Wiglenda2, Alexander Buntru2,HannaWolf1, Stephanie Weber1,InaVorberg 1, Alina Dagane2, Gunnar Dittmar2,3,ErichWanker2, Dan Ehninger1 and Sybille Krauss1 Abstract Alzheimer’s disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-β (Aβ) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aβ peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aβ in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These 1234567890 1234567890 findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.
[Show full text]
Distinct Interactions of Eif4a and Eif4e with RNA Helicase Ded1 Stimulate Translation in Vivo Suna Gulay, Neha Gupta, Jon R Lorsch, Alan G Hinnebusch*
RESEARCH ARTICLE Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo Suna Gulay, Neha Gupta, Jon R Lorsch, Alan G Hinnebusch* Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States Abstract Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5’UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1- CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5’UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G. Introduction Eukaryotic translation initiation is an intricate process that ensures accurate selection and decoding *For correspondence: of the mRNA start codon. Initiation
[Show full text]
Lncrna SNHG8 Is Identified As a Key Regulator of Acute Myocardial
Zhuo et al. Lipids in Health and Disease (2019) 18:201 https://doi.org/10.1186/s12944-019-1142-0 RESEARCH Open Access LncRNA SNHG8 is identified as a key regulator of acute myocardial infarction by RNA-seq analysis Liu-An Zhuo, Yi-Tao Wen, Yong Wang, Zhi-Fang Liang, Gang Wu, Mei-Dan Nong and Liu Miao* Abstract Background: Long noncoding RNAs (lncRNAs) are involved in numerous physiological functions. However, their mechanisms in acute myocardial infarction (AMI) are not well understood. Methods: We performed an RNA-seq analysis to explore the molecular mechanism of AMI by constructing a lncRNA-miRNA-mRNA axis based on the ceRNA hypothesis. The target microRNA data were used to design a global AMI triple network. Thereafter, a functional enrichment analysis and clustering topological analyses were conducted by using the triple network. The expression of lncRNA SNHG8, SOCS3 and ICAM1 was measured by qRT-PCR. The prognostic values of lncRNA SNHG8, SOCS3 and ICAM1 were evaluated using a receiver operating characteristic (ROC) curve. Results: An AMI lncRNA-miRNA-mRNA network was constructed that included two mRNAs, one miRNA and one lncRNA. After RT-PCR validation of lncRNA SNHG8, SOCS3 and ICAM1 between the AMI and normal samples, only lncRNA SNHG8 had significant diagnostic value for further analysis. The ROC curve showed that SNHG8 presented an AUC of 0.850, while the AUC of SOCS3 was 0.633 and that of ICAM1 was 0.594. After a pairwise comparison, we found that SNHG8 was statistically significant (P SNHG8-ICAM1 = 0.002; P SNHG8-SOCS3 = 0.031).
[Show full text]
Nat1 Promotes Translation of Specific Proteins That Induce Differentiation of Mouse Embryonic Stem Cells
Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells Hayami Sugiyamaa, Kazutoshi Takahashia,b, Takuya Yamamotoa,c,d, Mio Iwasakia, Megumi Naritaa, Masahiro Nakamuraa, Tim A. Randb, Masato Nakagawaa, Akira Watanabea,c,e, and Shinya Yamanakaa,b,1 aDepartment of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; bGladstone Institute of Cardiovascular Disease, San Francisco, CA 94158; cInstitute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8501, Japan; dJapan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo 100-0004, Japan; and eJapan Science and Technology Agency (JST)-CREST, Saitama 332-0012, Japan Contributed by Shinya Yamanaka, November 22, 2016 (sent for review October 18, 2016; reviewed by Katsura Asano and Keisuke Kaji) Novel APOBEC1 target 1 (Nat1) (also known as “p97,”“Dap5,” and To study the role of Nat1 in cell differentiation further, we “Eif4g2”) is a ubiquitously expressed cytoplasmic protein that is homol- generated mouse embryonic stem cells (mES cells) lacking both ogous to the C-terminal two thirds of eukaryotic translation initiation alleles of the Nat1 gene. mES cells were derived from blastocysts factor 4G (Eif4g1). We previously showed that Nat1-null mouse embry- in 1981 (11, 12) and possess two unique properties. First, ES cells onic stem cells (mES cells) are resistant to differentiation. In the current have the potential to self-renew indefinitely (maintenance). Sec- study, we found that NAT1 and eIF4G1 share many binding proteins, ond, ES cells have the potential to differentiate into all somatic such as the eukaryotic translation initiation factors eIF3 and eIF4A and and germ cell types (pluripotency) that make up the body.
[Show full text]
Diagnostic Yield and Novel Candidate Genes by Exome Sequencing in 152 Consanguineous Families with Neurodevelopmental Disorders
Research JAMA Psychiatry | Original Investigation Diagnostic Yield and Novel Candidate Genes by Exome Sequencing in 152 Consanguineous Families With Neurodevelopmental Disorders Miriam S. Reuter, MD; Hasan Tawamie, MA; Rebecca Buchert, MA; Ola Hosny Gebril, MD; Tawfiq Froukh, PhD; Christian Thiel, MD; Steffen Uebe, PhD; Arif B. Ekici, PhD; Mandy Krumbiegel, PhD; Christiane Zweier, MD; Juliane Hoyer, MD; Karolin Eberlein, MD; Judith Bauer, MD; Ute Scheller, MD; Tim M. Strom, MD; Sabine Hoffjan, MD; Ehab R. Abdelraouf, MD; Nagwa A. Meguid, MD, PhD; Ahmad Abboud, MD; Mohammed Ayman Al Khateeb, MD; Mahmoud Fakher, MD; Saber Hamdan, MD; Amina Ismael, MD; Safia Muhammad, MD; Ebtessam Abdallah, MD, PhD; Heinrich Sticht, PhD; Dagmar Wieczorek, MD; André Reis, MD; Rami Abou Jamra, MD Supplemental content IMPORTANCE Autosomal recessive inherited neurodevelopmental disorders are highly heterogeneous, and many, possibly most, of the disease genes are still unknown. OBJECTIVES To promote the identification of disease genes through confirmation of previously described genes and presentation of novel candidates and provide an overview of the diagnostic yield of exome sequencing in consanguineous families. DESIGN, SETTING, AND PARTICIPANTS Autozygosity mapping in families and exome sequencing of index patients were performed in 152 consanguineous families (the parents descended from a same ancestor) with at least 1 offspring with intellectual disability (ID). The study was conducted from July 1, 2008, to June 30, 2015, and data analysis was conducted from July 1, 2015, to August 31, 2016. RESULTS Of the 152 consanguineous families enrolled, 1 child (in 45 families [29.6%]) or multiple children (107 families [70.4%]) had ID; additional features were present in 140 of the families (92.1%).
[Show full text]
UNDERSTANDING the ROLE of EIF4A in GENE REGULATION in HEALTH and DISEASE THOMAS EDWARD WEBB B.Sc
UNDERSTANDING THE ROLE OF EIF4A IN GENE REGULATION IN HEALTH AND DISEASE THOMAS EDWARD WEBB B.Sc. (Hons, Warwick) M.Sc. (Oxon) Thesis submitted to The University of Nottingham for the degree of Doctor of Philosophy (July 2012) 1 Abstract eIF4A Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase responsible for unwinding the secondary structure of mRNAs. In humans, eIF4A exists as three separate paralogs: eIF4AI and eIF4AII possess a high degree of homology while eIF4AIII is distinct. Knockdown of eIF4AII had no effect on the expression of a reporter construct containing a structured RNA hairpin. Knockdown of eIF4AI and treatment with hippuristanol (an eIF4A inhibitor) caused a dramatic reduction in the hairpin-mediated gene. This reporter system was developed as part of this project to act as a screen for eIF4A activity along with an in vitro screening approach. PDCD4 The activity of eIF4A is suppressed in vivo by the tumour suppressor PDCD4. The fact that loss of PDCD4 function increases the severity of DNA damage is probably attributable its eIF4A-suppressive activity. Alzheimer's Disease Based on previous microarray data, it was supposed that eIF4A inhibition may be therapeutically beneficial in the treatment of Alzheimer's disease. As part of this project, it was demonstrated that eIF4A suppression significantly reduced the expression of reporter genes preceded by the 5’ UTRs of genes predicted to play harmful roles in Alzheimer’s disease. The expression of reporter genes preceded by the 5’ UTR sequences of genes predicted to be beneficial in Alzheimer's were not affected by this suppression.
[Show full text]
Viewed and Published Immediately Upon Acceptance Cited in Pubmed and Archived on Pubmed Central Yours — You Keep the Copyright
Molecular Cancer BioMed Central Research Open Access Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling Ingo Nindl*1, Chantip Dang1, Tobias Forschner1, Ralf J Kuban2, Thomas Meyer3, Wolfram Sterry1 and Eggert Stockfleth1 Address: 1Department of Dermatology, Charité, Skin Cancer Center Charité, University Hospital of Berlin, Charitéplatz 1, D-10117 Berlin, Germany, 2Institute of Biochemistry, Charité, University Hospital of Berlin, Monbijoustr. 2, D-10098 Berlin, Germany and 3Institut of Pathology and Molecularbiology (IPM), Lademannbogen 61, D-22339 Hamburg, Germany Email: Ingo Nindl* - [email protected]; Chantip Dang - [email protected]; Tobias Forschner - [email protected]; Ralf J Kuban - [email protected]; Thomas Meyer - [email protected]; Wolfram Sterry - [email protected]; Eggert Stockfleth - [email protected] * Corresponding author Published: 08 August 2006 Received: 15 February 2006 Accepted: 08 August 2006 Molecular Cancer 2006, 5:30 doi:10.1186/1476-4598-5-30 This article is available from: http://www.molecular-cancer.com/content/5/1/30 © 2006 Nindl et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Carcinogenesis is a multi-step process indicated by several genes up- or down- regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). Results: Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed.
[Show full text]