Relax, Don’T RAN Translate It

Total Page:16

File Type:pdf, Size:1020Kb

Relax, Don’T RAN Translate It Neuron Previews Relax, Don’t RAN Translate It Katherine M. Wilson,1,2 Bhavana Muralidharan,1,2,3 and Adrian M. Isaacs1,2,* 1UK Dementia Research Institute at UCL, Gower Street, London WC1E 6BT, UK 2Department of Neurodegenerative Disease, Queen Square UCL Institute of Neurology, London WC1N 3BG, UK 3Brain Development and Disease Mechanisms, Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India, 560065 *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2019.11.014 The (GGGGCC)n repeat expansion in C9orf72, which is the most common cause of frontotemporal dementia and amyotrophic lateral sclerosis, is translated through repeat-associated non-AUG (RAN) translation. In this issue of Neuron, Cheng et al. (2019) report that the helicase DDX3X, which unwinds (or relaxes) RNA, sup- presses RAN translation and toxicity. The frontotemporal dementia (FTD)- and port, the proteasome complex, RNA bind- These results point toward a specific amyotrophic lateral sclerosis (ALS)-caus- ing, and helicases. To validate hits from role of DDX3X in repeat RNA translation. ative (GGGGCC)n repeat expansion in the initial screen, a dual luciferase cell The RNA helicase activity of DDX3X is intron 1 of C9orf72 is proposed to drive line was used in which cells express dependent on ATPase activity. To ascer- toxicity through several modes of action NanoLuciferase (NanoLuc) via RAN trans- tain whether ATPase activity was required (Balendra and Isaacs, 2018). One poten- lation as well as firefly luciferase (FLuc) via for the effect of DDX3X on DPR levels, tial mechanism is production of dipeptide canonical (AUG) translation. 48 genes wild-type DDX3X and ATPase-defective repeat proteins (DPRs) by repeat-associ- were re-tested in this secondary screen, mutant forms of DDX3X were overex- ated non-AUG (RAN) translation of the and knockdown of the RNA helicase pressed in cells. Only the wild-type expanded repeats. This non-canonical DDX3X caused the greatest change in DDX3X could lower DPR levels, suggest- translation occurs in all three reading NanoLuc levels, increasing RAN transla- ing a dependence on ATPase activity for frames, in both the sense and antisense tion by more than 2-fold while slightly modulation of RAN translation. Recombi- directions, leading to the production of reducing AUG-FLuc translation. DDX3X nant DDX3X was then used to show direct five different DPRs: poly-GA, poly-GP, is a conserved DEAD-box RNA helicase binding of repeat RNA to DDX3X and that and poly-GR from sense transcripts and shown to be involved in RNA transcrip- GGGGCC repeat RNA could stimulate poly-GP, poly-PR, and poly-AP from anti- tion, splicing, export, and translation, as DDX3X ATPase activity. sense transcripts. One possible therapeu- well as having roles in cell cycle, tumori- Following on from this cell line and tic intervention could be to reduce RAN genesis, and stress granule formation in vitro data, Cheng et al. (2019) translation. Yet, the mechanism by which (Linder and Jankowsky, 2011). Addition- next investigated whether DDX3X modu- RAN translation occurs is still not fully un- ally, loss-of-function mutations in DDX3X lation could affect RAN-translation- derstood. Targeted genetic screens have have been identified as a cause of induced toxicity in vivo. Here, they started to identify genes that regulate X-linked recessive intellectual disability utilized a Drosophila model expressing RAN translation (Goodman et al., 2019; (Snijders Blok et al., 2015). Knockdown (GGGGCC)58 in the fly eye, which causes Linsalata et al., 2019; Yamada et al., of DDX3X caused an increase in RAN- mild eye degeneration. Expression of 2019). In this issue of Neuron, Cheng translated NanoLuc signal in all three either mutant belle (the Drosophila et al. (2019) now report the first genome- sense frames; however, no change in DDX3X homolog) or belle RNAi knock- wide screen for modulators of C9orf72 antisense RAN translation was observed, down exacerbated the eye degeneration RAN translation. They first created a re- indicating specificity for sense GGGGCC phenotype and led to increased poly-GP porter cell line expressing the C9orf72 in- repeats. The increase in DPR levels was levels. These results suggest that DDX3X tronic sequence directly upstream of the also observed when in vitro transcribed may regulate RAN translation and associ- repeats, followed by (GGGGCC)70 and GGGGCC repeat-NanoLuc RNA was ated toxicity in vivo. Importantly, in addi- an eGFP tag in the poly-GA frame. Using transfected into DDX3X knockdown cells, tion to these overexpression studies, this reporter cell line, a genome-wide indicating an effect on RAN translation Cheng et al. (2019) next investigated the CRISPR-Cas9 knockout screen was car- and not transcription. Polysome fraction- effect of DDX3X on endogenous RAN ried out using fluorescence-activated ation revealed a striking change in distri- translation in C9orf72 patient cells. In cell sorting to monitor changes in poly- bution of repeat RNA, from monosomes both C9orf72 patient lymphoblasts and GA-eGFP levels. This identified 76 in controls to translating polysomes in induced pluripotent stem cells (iPSCs), enhancer and 145 suppressor genes. DDX3X siRNA-treated cells. No change levels of poly-GP were greatly increased Gene Ontology analysis identified an in the polysome distribution of the AUG- by knockdown of DDX3X. All these data enrichment in genes involved in pathways FLuc RNA was observed, and no major in various systems, including patient cells, relevant to protein translation and degra- changes in global translation were identi- point to a role for DDX3X in regulating dation: translation initiation, RNA trans- fied using puromycin incorporation. RAN translation of C9orf72 GGGGCC Neuron 104, December 4, 2019 ª 2019 Elsevier Inc. 827 Neuron Previews result is that the region immediately up- stream of the FMR1 CGG repeats is also GC rich and that DDX3X activity is required to unwind this sequence in order to allow the PIC access to the repeats. Therefore, the requirement to unwind the upstream sequence trumps the need to maintain the secondary structure of the repeats themselves. Some unwinding of the repeats is presumably needed to allow translation to proceed after initiation has occurred. Consequently, a complex balance of maintaining and resolving repeat structure may ultimately be required for effective RAN translation. What is clear is that the effects of DDX3X are both sequence and context specific. It may also be challenging to directly target DDX3X therapeutically Figure 1. Loss of DDX3X RNA Helicase Activity Increases C9orf72 Repeat RAN Translation given its diverse roles in the cell, which The (GGGGCC)n repeat expansion in C9orf72 can be translated via repeat-associated non-AUG (RAN) suggests a relatively broad range of translation in each reading frame to produce poly-GA, poly-GP, and poly-GR dipeptide repeat proteins (DPRs). The RNA helicase activity of DDX3X reduces (GGGGCC)n RAN translation. Conversely, upon loss targets, but further work will determine of DDX3X, the repeat RNA forms secondary structures that act as IRES elements and/or stall scanning whether specificity can be achieved. In ribosome complexes, leading to increased RAN translation and DPR levels. summary, the innovative genome-wide CRISPR/Cas9 screen described here repeats, as knockdown of DDX3X consis- translation in vitro using a (CGG)100 Nano- has identified new modifiers of GGGGCC tently increases DPR levels. However, for Luc assay and improved survival of pri- repeat RAN translation, which help shed there to be therapeutic relevance, the mary rat neurons expressing (CGG)100. new light on this mysterious form of un- opposite also needs to be demon- Thus, they conclude that DDX3X knock- conventional translation. strated—that overexpression of DDX3X down suppresses RAN translation and can reduce DPR levels. Cheng et al. associated toxicity. How can DDX3X REFERENCES (2019) address this question in two sys- have opposing effects on RAN translation tems. First, they report that overexpres- of C9orf72 and FMR1 expanded repeats? Balendra, R., and Isaacs, A.M. (2018). C9orf72- mediated ALS and FTD: multiple pathways to dis- sion of DDX3X decreases poly-GP in their In the first pioneering description of ease. Nat. Rev. Neurol. 14, 544–558. dual luciferase assay. Second, they show RAN translation it was shown that the sec- that DDX3X overexpression can reduce ondary structure of the repeat RNA plays Cheng, W., Wang, S., Mestre, A.A., Fu, C., Makarem, A., Xian, F., Hayes, L.R., Lopez- poly-GP in C9orf72 patient iPSC-derived an important role. Two non-mutually Gonzalez, R., Drenner, K., Jiang, J., et al. (2018). motor neurons. Impressively, this reduc- exclusive mechanisms have been sug- C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2a tion was sufficient to reduce both gested to initiate RAN translation: cap- phosphorylation. Nat. Commun. 9,51. the increased sensitivity to glutamate- dependent scanning of the pre-initiation induced cell death and the disrupted nu- complex (PIC) and cap-independent utili- Cheng, W., Wang, S., Zhang, Z., Morgens, D.W., Hayes, L.R., Lee, S., Portz, B., Xie, Y., Nguyen, cleocytoplasmic transport observed in zation of an internal ribosome entry site B.V., Haney, M.S., et al. (2019). CRISPR-Cas9 C9orf72 patient iPSC neurons. (IRES) (Cheng et al., 2018; Green et al., Screens Identify the RNA Helicase DDX3X as a It is an attractive model in which RAN 2017; Tabet et al., 2018). RNA secondary Repressor of C9ORF72 (GGGGCC)n Repeat- Associated Non-AUG Translation. Neuron 104, translation is regulated by specific factors structure is relevant for both mechanisms, this issue, 885–898. and mechanisms. This would allow for as it is known to be important for IRES- Goodman, L.D., Prudencio, M., Srinivasan, A.R., specific targeting of RAN translation mediated translation, and repeat RNA Rifai, O.M., Lee, V.M.Y., Petrucelli, L., and Bonini, in multiple repeat expansion diseases.
Recommended publications
  • Wnt Signalling in Melanoma and Ageing
    MINIREVIEW British Journal of Cancer (2016) 115, 1273–1279 | doi: 10.1038/bjc.2016.332 Keywords: Wnt; metastasis; melanoma; ageing; sFRP2; Wnt5a In the Wnt-er of life: Wnt signalling in melanoma and ageing Amanpreet Kaur1,2, Marie R Webster1 and Ashani T Weeraratna*,1 1Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA and 2University of the Sciences, Philadelphia, PA, USA Although the clinical landscape of melanoma is improving rapidly, metastatic melanoma remains a deadly disease. Age remains one of the greatest risk factors for melanoma, and patients older than 55 have a much poorer prognosis than younger individuals, even when the data are controlled for grade and stage. The reasons for this disparity have not been fully uncovered, but there is some recent evidence that Wnt signalling may have a role. Wnt signalling is known to have roles both in cancer progression as well as in organismal ageing. In melanoma, the interplay of Wnt signalling pathways is complex, with different members of the Wnt family guiding different aspects of invasion and proliferation. Here, we will briefly review the current literature addressing the roles of different Wnt pathways in melanoma pathogenesis, provide an overview of Wnt signalling during ageing, and discuss the intersection between melanoma and ageing in terms of Wnt signalling. AGE IS A PROGNOSTIC FACTOR FOR MELANOMA also contribute to the age-induced progression of cancer. For example, it has long been proposed that the ageing stroma As human lifespan increases, there is a growing concern over the contributes to cancer progression, based on the studies using availability of treatments to manage the increasing incidence of senescence as an artificial model of ageing (Campisi, 2013; Campisi cancer in aged individuals.
    [Show full text]
  • Ddx3x Mouse Shrna Lentiviral Particle (Locus ID 13205) – TL519041V | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TL519041V Ddx3x Mouse shRNA Lentiviral Particle (Locus ID 13205) Product data: Product Type: shRNA Lentiviral Particles Product Name: Ddx3x Mouse shRNA Lentiviral Particle (Locus ID 13205) Locus ID: 13205 Synonyms: D1Pas1-rs2; Ddx3; Fin14 Vector: pGFP-C-shLenti (TR30023) Format: Lentiviral particles RefSeq: NM_010028, NM_010028.1, NM_010028.2, NM_010028.3, BC172016, BC067210, BC083059, BC150862 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 Ddx3x Mouse shRNA Lentiviral Particle (Locus ID 13205) – TL519041V Summary: Multifunctional ATP-dependent RNA helicase. The ATPase activity can be stimulated by various ribo- and deoxynucleic acids indicative for a relaxed substrate specificity. In vitro can unwind partially double-stranded DNA with a preference for 5'-single-stranded DNA overhangs. Is involved in several steps of gene expression, such as transcription, mRNA maturation, mRNA export and translation. However, the exact mechanisms are not known and some functions may be specific for a subset of mRNAs. Involved in transcriptional regulation. Can enhance transcription from the CDKN1A/WAF1 promoter in a SP1-dependent manner. Found associated with the E-cadherin promoter and can down-regulate transcription from the promoter. Involved in regulation of translation initiation. Proposed to be involved in positive regulation of translation such as of cyclin E1/CCNE1 mRNA and specifically of mRNAs containing complex secondary structures in their 5'UTRs; these functions seem to require RNA helicase activity.
    [Show full text]
  • DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy
    cells Review DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy Lu Zhang 1,2 and Xiaogang Li 2,3,* 1 Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060, China; [email protected] 2 Department of Internal Medicine, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA * Correspondence: [email protected]; Tel.: +1-507-266-0110 Abstract: Cell cycle is regulated through numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest, or undergo apoptosis. Abnormal cell cycle regula- tion has been linked to many diseases. Thus, there is an urgent need to understand the diverse molecular mechanisms of how the cell cycle is controlled. RNA helicases constitute a large family of proteins with functions in all aspects of RNA metabolism, including unwinding or annealing of RNA molecules to regulate pre-mRNA, rRNA and miRNA processing, clamping protein complexes on RNA, or remodeling ribonucleoprotein complexes, to regulate gene expression. RNA helicases also regulate the activity of specific proteins through direct interaction. Abnormal expression of RNA helicases has been associated with different diseases, including cancer, neurological disorders, aging, and autosomal dominant polycystic kidney disease (ADPKD) via regulation of a diverse range of cellular processes such as cell proliferation, cell cycle arrest, and apoptosis. Recent studies showed that RNA helicases participate in the regulation of the cell cycle progression at each cell cycle phase, including G1-S transition, S phase, G2-M transition, mitosis, and cytokinesis.
    [Show full text]
  • Antiviral Potential Coupled to Genome Stability: the Multifaceted Roles of DDX3X Protein
    Dipartimento di Biologia e Biotecnologie “L. Spallanzani” Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare Luigi Luca Cavalli-Sforza Antiviral potential coupled to genome stability: the multifaceted roles of DDX3X protein Valentina Riva Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare Ciclo XXXII – A.A. 2016-2019 Dipartimento di Biologia e Biotecnologie “L. Spallanzani” Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare Luigi Luca Cavalli-Sforza Antiviral potential coupled to genome stability: the multifaceted roles of DDX3X protein Valentina Riva Supervised by Prof. Giovanni Maga Dottorato di Ricerca in Genetica, Biologia Molecolare e Cellulare Ciclo XXXII – A.A. 2016-2019 Abstract The human RNA helicase DDX3X is a real multifaceted enzyme. Like all the other DEAD-box proteins of the same family, DDX3X participates into different steps of RNA metabolism. Moreover, DDX3X is one of the actors of cell cycle regulation, innate immunity and apoptosis processes. Our group started to look at DDX3X as an interesting protein since it has primary roles in viral infections and tumor development too. In the context of viral infections, DDX3X possesses dual roles: it acts as an antiviral or proviral factor regulating viral replication at different levels (regulation of genome duplication and/or gene expression and host innate immunity activation). From these observations, it came the idea to use DDX3X as a possible therapeutic target to inhibit a function essential for the viral replication, but dispensable for the human cell. In collaboration with the University of Siena (Prof. Maurizio Botta), we developed some inhibitor molecules able to recognize two different DDX3X pockets: the helicase binding pocket and the unique motif of DDX3X.
    [Show full text]
  • Frequent Somatic Mutations in Components of the RNA Processing Machinery in Chronic Lymphocytic Leukemia
    Letters to the Editor 1600 CONFLICT OF INTEREST 2 Sokol L, Loughran TP Jr. Large granular lymphocyte leukemia. Oncologist 2006; 11: WK, CH, TH and SS are part owners of the MLL Munich Leukemia Laboratory. AF and 263–273. VG are employed by the MLL Munich Leukemia Laboratory. 3 Koskela HL, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanmaki H, Andersson EI et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012; 366: 1905–1913. ACKNOWLEDGEMENTS 4 Jerez A, Clemente MJ, Makishima H, Koskela H, Leblanc F, Ng KP et al. STAT3 mutations unify the pathogenesis of chronic lymphoproliferative dis- We thank all clinicians for sending samples to our laboratory for diagnostic purposes, orders of NK cells and T cell large granular lymphocyte leukemia. Blood 2012; 120: and for providing clinical information and follow-up data. In addition, we would like 3048–3057. to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching 5 Kern W, Bacher U, Haferlach C, Alpermann T, Dicker F, Schnittger S et al. together many aspects in the field of leukemia diagnostics and research. In addition, Frequency and prognostic impact of the aberrant CD8 expression in 5,523 we are grateful for the data management support performed by Tamara Alpermann. patients with chronic lymphocytic leukemia. Cytometry B Clin Cytom 2012; 82: 145–150. A Fasan, W Kern, V Grossmann, C Haferlach, 6 van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL T Haferlach and S Schnittger et al. Design and standardization of PCR primers and protocols for detection of MLL Munich Leukemia Laboratory, Munich, Germany clonal immunoglobulin and T-cell receptor gene recombinations in suspect lym- E-mail: [email protected] phoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.
    [Show full text]
  • By Submitted in Partial Satisfaction of the Requirements for Degree of in in the GRADUATE DIVISION of the UNIVERSITY of CALIFORN
    DDX3X and C12ORF57/Camkinin: Insight into the function and malfunction of two genes implicated in agenesis of the corpus callosum by Ruiji Jiang DISSERTATION Submitted in partial satisfaction of the requirements for degree of DOCTOR OF PHILOSOPHY in Biomedical Sciences in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Approved: ______________________________________________________________________________Aimee Kao Chair ______________________________________________________________________________Elliott Sherr ______________________________________________________________________________John Rubenstein ______________________________________________________________________________Kevin Shannon ______________________________________________________________________________ Committee Members ii Acknowledgements I would like to thank Malek Chouchane and Erik Ullian for their assistance in planning and collecting the electrophysiology data for the sections dealing with C12ORF57. The Linda Richards lab at the Queensland Brain Insitute, specifically Ching Moey and also provided valuable support in performing dual stain in situs and immunohistochemistry for the cell type specific expression assays as well as sectioning for mouse brains. Thanks to Suling Wong for her artistic expertise in illustrating our C12ORF57/Camkinin model Stephen Floor and the Doudna lab at Berkeley provided invaluable assistance in explaining the principles of the DDX3X unwinding assay, as well as providing technical assistance in purifying protein,
    [Show full text]
  • Quantitative Analysis of Y-Chromosome Gene Expression Across 36 Human Tissues 6 7 8 9 Alexander K
    Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press 1 2 3 4 5 Quantitative analysis of Y-Chromosome gene expression across 36 human tissues 6 7 8 9 Alexander K. Godfrey1,2, Sahin Naqvi1,2, Lukáš Chmátal1, Joel M. Chick3, 10 Richard N. Mitchell4, Steven P. Gygi3, Helen Skaletsky1,5, David C. Page1,2,5* 11 12 13 1 Whitehead Institute, Cambridge, MA, USA 14 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 15 3 Department of Cell Biology, Harvard Medical School, Boston, MA, USA 16 4 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 17 5 Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA, USA 18 19 20 21 *corresponding author: 22 Email: [email protected] 23 24 25 Running title: 26 Human Y-Chromosome gene expression in 36 tissues 27 28 29 Keywords: 30 Y Chromosome, sex chromosomes, sex differences, EIF1AY, EIF1AX 31 Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press 32 ABSTRACT 33 Little is known about how human Y-Chromosome gene expression directly contributes to 34 differences between XX (female) and XY (male) individuals in non-reproductive tissues. Here, 35 we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues 36 from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly 37 expressed outside the testis, we report many instances of elevated Y-Chromosome gene 38 expression in a non-reproductive tissue.
    [Show full text]
  • The Role of Melanocyte Lineage Genes in Melanoma Sanna, Adriana
    The Role of Melanocyte Lineage Genes in Melanoma Sanna, Adriana 2020 Document Version: Publisher's PDF, also known as Version of record Link to publication Citation for published version (APA): Sanna, A. (2020). The Role of Melanocyte Lineage Genes in Melanoma. Lund University, Faculty of Medicine. Total number of authors: 1 General rights Unless other specific re-use rights are stated the following general rights apply: Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Read more about Creative commons licenses: https://creativecommons.org/licenses/ Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 ADRIANA SANNA ADRIANA About this Thesis Melanocyteof The Role Lineage Genes Melanomain Malignant melanoma is the deadliest form of skin cancer, and the number of cases is escalating worldwide. In spite of the breakthrough in therapeutics The Role of Melanocyte made available to melanoma patients in the last decades, treatment resistance followed by tumor relapse remains the leading cause of patient death.
    [Show full text]
  • A Novel JAK1 Mutant Breast Implant-Associated Anaplastic Large Cell Lymphoma Patient-Derived Xenograft Fostering Pre- Clinical Discoveries
    Cancers 2019 S1 of S18 Supplementary Materials: A Novel JAK1 Mutant Breast Implant-Associated Anaplastic Large Cell Lymphoma Patient-Derived Xenograft Fostering Pre- Clinical Discoveries Danilo Fiore, Luca Vincenzo Cappelli, Paul Zumbo, Jude M. Phillip, Zhaoqi Liu, Shuhua Cheng, Liron Yoffe, Paola Ghione, Federica Di Maggio, Ahmet Dogan, Inna Khodos, Elisa de Stanchina, Joseph Casano, Clarisse Kayembe, Wayne Tam, Doron Betel, Robin Foa’, Leandro Cerchietti, Raul Rabadan, Steven Horwitz, David M. Weinstock and Giorgio Inghirami A B C Figure S1. (A) Histology micrografts on IL89 PDTX show overall similarity between T1 T3 and T7 passages (upper panels). Immunohistochemical stains with the indicated antibodies (anti-CD3, anti- CD25 and anti-CD8 [x20]) (lower panels). (B) Flow cytometry panel comprehensive of the most represented surface T-cell lymphoma markers, including: CD2, CD3, CD4, CD5, CD8, CD16, CD25, CD30, CD56, TCRab, TCRgd. IL89 PDTX passage T3 is here depicted for illustration purposes. (C) Analysis of the TCR gamma specific rearrangement clonality in IL89 diagnostic sample and correspondent PDTX after 1 and 5 passages (T1 and T5). A WT Primary p.G1097D IL89 T1 p.G1097D IL89 T5 p.G1097D IL89 cell line B Figure S2. (A) Sanger sequencing confirms the presence of the JAK1 p.G1097D mutation in IL89 PDTX samples and in the cell line, but the mutation is undetectable in the primary due to the low sensitivity of the technique. (B) Manual backtracking of mutations in the primary tumor using deep sequencing data allowed for the identification of several hits at a very low VAF compared to the PDTX-T5. A B IL89 CTRL 30 CTRL Ruxoli?nib S 20 M Ruxoli?nib A R G 10 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 1 1 1 1 1 WEEKS AFTER ENGRAFTMENT Figure S3.
    [Show full text]
  • DDX3X-Related Neurodevelopmental Disorder
    NLM Citation: Johnson-Kerner B, Snijders Blok L, Suit L, et al. DDX3X- Related Neurodevelopmental Disorder. 2020 Aug 27. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2020. Bookshelf URL: https://www.ncbi.nlm.nih.gov/books/ DDX3X-Related Neurodevelopmental Disorder Bethany Johnson-Kerner, MD, PhD,1 Lot Snijders Blok, MD,2 Lindsey Suit, BA,1 Julian Thomas, MD,3 Tjitske Kleefstra, MD, PhD,4 and Elliott H Sherr, MD, PhD5 Created: August 27, 2020. Summary Clinical characteristics DDX3X-related neurodevelopmental disorder (DDX3X-NDD) typically occurs in females and very rarely in males. All affected individuals reported to date have developmental delay / intellectual disability (ID) ranging from mild to severe; about 50% of affected girls remain nonverbal after age five years. Hypotonia, a common finding, can be associated with feeding difficulty in infancy. Behavioral issues can include autism spectrum disorder, attention-deficit/hyperactivity disorder and hyperactivity, self-injurious behavior, poor impulse control, and aggression. Other findings can include seizures, movement disorders (dyskinesia, spasticity, abnormal gait), vision and hearing impairment, congenital heart defects, respiratory difficulties, joint laxity, and scoliosis. Neuroblastoma has been observed in three individuals. Diagnosis/testing The diagnosis of DDX3X-NDD is established in a female proband with suggestive findings and a heterozygous de novo DDX3X pathogenic variant identified by molecular genetic testing and in a male proband with suggestive findings and a hemizygous DDX3X pathogenic variant. Management Treatment of manifestations: Treatment is symptomatic and focuses on optimizing the individual's abilities using a multidisciplinary approach that should also include psychosocial support for family members.
    [Show full text]
  • DDX3 / DDX3X Antibody (N-Terminus) Rabbit Polyclonal Antibody Catalog # ALS12227
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 DDX3 / DDX3X Antibody (N-Terminus) Rabbit Polyclonal Antibody Catalog # ALS12227 Specification DDX3 / DDX3X Antibody (N-Terminus) - Product Information Application ICC, IF, WB Primary Accession O00571 Reactivity Human, Mouse Host Rabbit Clonality Polyclonal Calculated MW 73kDa KDa DDX3 / DDX3X Antibody (N-Terminus) - Additional Information Gene ID 1654 Other Names Immunocytochemistry of DDX3 in HepG2 ATP-dependent RNA helicase DDX3X, cells with DDX3 antibody at 10 ug/ml. 3.6.4.13, DEAD box protein 3, X-chromosomal, DEAD box, X isoform, Helicase-like protein 2, HLP2, DDX3X, DBX, DDX3 Target/Specificity 16 amino acid peptide from near the amino terminus of human DDX3. Reconstitution & Storage Short term 4°C, long term aliquot and store at -20°C, avoid freeze thaw cycles. Store undiluted. Precautions DDX3 / DDX3X Antibody (N-Terminus) is for Immunofluorescence of DDX3 in HepG2 cells research use only and not for use in with DDX3 antibody at 20 ug/ml. diagnostic or therapeutic procedures. DDX3 / DDX3X Antibody (N-Terminus) - Protein Information Name DDX3X Synonyms DBX {ECO:0000303|PubMed:15294876}, DDX3 Function Multifunctional ATP-dependent RNA helicase Page 1/8 10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 (PubMed:<a href="http://www.uniprot.org/c itations/17357160" target="_blank">17357160</a>, PubMed:<a href="http://www.uniprot.org/ci tations/21589879" target="_blank">21589879</a>, PubMed:<a href="http://www.uniprot.org/ci tations/31575075" target="_blank">31575075</a>). The ATPase activity can be stimulated by various ribo-and deoxynucleic acids indicative for a relaxed substrate specificity (PubMed:<a href="http://www.uniprot.org/c itations/29222110" target="_blank">29222110</a>).
    [Show full text]
  • Genome-Wide Analysis of Translational Efficiency Reveals Distinct but Overlapping Functions of Yeast DEAD-Box RNA Helicases Ded1 and Eif4a
    Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Research Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A Neelam Dabas Sen,1 Fujun Zhou,1 Nicholas T. Ingolia,2 and Alan G. Hinnebusch1 1Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA; 2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint pro- filing. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more perva- sive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs.
    [Show full text]