scholarly journals Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000920
Author(s):  
Abdalla Akef ◽  
Kathy McGraw ◽  
Steven D. Cappell ◽  
Daniel R. Larson
Keyword(s):  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Common Mutation ◽  
Small Nuclear Rna ◽  
Splice Site Selection ◽  
Downstream Effector ◽  
Heterodimeric Complex ◽  
Auxiliary Factor ◽  
Mutant Cells ◽  
Missense Substitution

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

scholarly journals Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation

2019 ◽  
Author(s):  
Abdalla Akef ◽  
Kathy McGraw ◽  
Steven D. Cappell ◽  
Daniel R. Larson
Keyword(s):  
Myeloid Leukemia ◽  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Common Mutation ◽  
Splice Site Selection ◽  
Downstream Effector ◽  
Processing Defects ◽  
Heterodimeric Complex ◽  
Global Increase ◽  
Missense Substitution

AbstractU2AF1 forms a heterodimeric complex with U2AF2 that is primarily responsible for 3’ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome and Acute Myeloid Leukemia, and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). However, the U2AF heterodimer also has a non-canonical function as a translational regulator. Here, we report that the U2AF1 S34F mutation results in specific mis-regulation of the translation initiation and ribosome biogenesis machinery, with the potential for widespread translational changes. The net result is a global increase in mRNA translation at the single cell level. Among the translationally upregulated targets of U2AF1-S34F are Nucleophosmin1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of wt/S34F cells and causes rRNA processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1 and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation which recapitulates translational mis-regulation in myeloid disease.

scholarly journals Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells

2017 ◽  
Vol 114 (30) ◽  
pp. E6117-E6126 ◽  
Author(s):  
Thomas C. J. Tan ◽  
John Knight ◽  
Thomas Sbarrato ◽  
Kate Dudek ◽  
Anne E. Willis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Ribosome Biogenesis ◽  
Cell Activation ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cell Receptor ◽  
Tcr Signaling

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

scholarly journals The DHX33 RNA Helicase Promotes mRNA Translation Initiation

2015 ◽  
Vol 35 (17) ◽  
pp. 2918-2931 ◽  
Author(s):  
Yandong Zhang ◽  
Jin You ◽  
Xingshun Wang ◽  
Jason Weber
Keyword(s):  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Rna Helicase ◽  
Rna Helicases ◽  
80S Ribosome ◽  
Protein Levels ◽  
Cell Growth And Proliferation ◽  
Rna Complexes

DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.

scholarly journals GCT-19. MODELING GERM CELL TUMORS WITH KIT MUTANT hiPSCs

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii331-iii331
Author(s):  
Sakura Kuzuoka ◽  
Yoji Kojima ◽  
Koichi Ichimura ◽  
Mitinori Saitou
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Germ Cell Tumors ◽  
Pediatric Brain Tumor ◽  
Common Mutation ◽  
Ras Pathway ◽  
Human Ipscs ◽  
Methylome Analysis ◽  
Mutant Lines ◽  
Mutant Cells

Abstract Central Nervous System Germ Cell Tumor (CNS GCT) is the second most common pediatric brain tumor in Japan, and within CNS GCT, germinoma is the most common subtype, accounting for 62.3%. Recent reports of transcriptome and methylome analysis suggested that germinoma highly resemble the state of gonocytes, the germ cells at around 5th to 7th week of human embryo development. It is also identified that 60% of germinoma harbored somatic mutations in KIT/RAS pathway. As the protocol to derive gonocytes from human iPSCs have been reported, we aimed to recapitulate tumorigenesis by generating human iPSCs bearing common genetic mutations and derive gonocytes from them. We first introduced the most common mutation KITD816V to human iPSCs using CRISPR/Cas9, and confirmed in iPSCs that mutated KIT was phosphorylated in the absence of ligand stimulation, and also found that KIT activation contribute to the phosphorylation of AKT but not of ERK. Upon differentiation towards primordial germ cell -like cells (PGCLCs), KIT mutant lines were efficiently induced into PGCLCs, however, by comparing conditions with or without KIT ligand (SCF), mutant lines exhibited less dependency to SCF compared to wildtype cells. Mutant cells were further differentiated to gonocytes following published protocol and the cells were collected for transcriptome analysis. By comparing with the transcriptome of germinoma, we confirmed that germinoma samples express germ cell genes similar to gonocytes. We are attempting to identify the molecular mechanism of tumorigenesis in relation to KIT activation using this system.

scholarly journals Ubiquitin proteomics reveals critical targets of the Nse1 RING domain in rDNA and genome stability

2021 ◽  
Author(s):  
Eva Ibars ◽  
Gemma Belli ◽  
Celia Casas ◽  
Joan Codina-Fabra ◽  
Marc Tarres ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Genome Stability ◽  
Ring Domain ◽  
Transcriptional Elongation ◽  
Label Free ◽  
Dna Damage Tolerance ◽  
Cellular Processes ◽  
Ubiquitin E3 Ligase ◽  
Polymerase I ◽  
Mutant Cells

Ubiquitination controls numerous cellular processes, and its deregulation is associated to many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and important functions in genome integrity. However, Nse1-dependent ubiquitin targets remain largely unknown. Here, we use label-free quantitative proteomics to analyse the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts on the ubiquitination of several proteins involved in DNA damage tolerance, ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of the Smc5/6 complex in chromosome segregation. In addition, our analysis uncovers an unexpected connection between Nse1 and RNA polymerase I (RNAP I) ubiquitination. Specifically, Nse1 promotes the ubiquitination of K408 and K410 in A190, the largest subunit of RNAP I, to induce its degradation. We propose that this mechanism contributes to Smc5/6-dependent rDNA disjunction in response to transcriptional elongation defects.

Identification of Critical Phosporylation Sites within NPM-ALK That Regulate JUNB mRNA Translation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3571-3571
Author(s):  
Claudia Fuchs ◽  
Paul Vesely ◽  
Isabella Bambach ◽  
Silvia Schauer ◽  
Werner Linkesch ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Translational Regulation ◽  
Chromosomal Translocation ◽  
Mrna Translation ◽  
Large Cell ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Anaplastic Lymphoma ◽  
Rps6 Phosphorylation ◽  
Mutant Cells

Abstract Anaplastic large cell lymphoma (ALCL) accounts for approximately 30% of childhood lymphomas and 3% of adult non-Hodgkin lymphomas. The nucleophosmin - anaplastic lymphoma kinase (NPM-ALK) fusion which is the product of a t(2;5)(p23;q35) chromosomal translocation is present in about half of nodal ALCL. Expression of this fusion kinase results in induction of the AP-1 transcription factor JunB and IL-3 independent outgrow of murine hematopoietic Ba/F3 cells. We demonstrated that wild type NPM-ALK increases the amount of ribosomes bound to JUNB mRNA resulting in its more effective translation in large polysomes. The NPM-ALK fusion tyrosine kinase has 20 potential tyrosine residues available for autophosphorylation and phosphorylation by other protein tyrosine kinases. Here we used series of Y-to-F-substituted mutants of NPM-ALK to identify tyrosine residues that are required to regulate the segregation of JUNB mRNAs between polysomes and monosomes as well as ribonucleic particles (RNPs). Neither JUNB transcription nor JunB translation was altered in Ba/F3 cells expressing NPM-ALK mutants Y17F/Y29F/Y67F Y138F/Y152F Y156F/Y191F/Y299F Y378F/Y418F/Y445F and Y646F/Y664F compared to NPM-ALK wild type. Conversely, in NPM-ALK Y567F/Y461F/Y644F mutant cells proliferation was markedly decreased. These cells demonstrated active MEK-ERK pathway, while AKT, mTOR, and rpS6 phosphorylation was impaired. Moreover a shift of JUNB mRNA from the polysomic to the monosomic/mRNP fraction could be observed. In conclusion, we identified specific NPM-ALK phosphorylation sites required to mediate the effect of NPM-ALK on the JUNB translational regulation and therefore provide further insights in the transforming mechanisms of the oncoprotein NPM-ALK.

scholarly journals Translation Stress Regulates Ribosome Synthesis and Cell Proliferation

2018 ◽  
Vol 19 (12) ◽  
pp. 3757 ◽  
Author(s):  
Sivakumar Vadivel Gnanasundram ◽  
Robin Fåhraeus
Keyword(s):  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Developmental Disorders ◽  
Mrna Translation ◽  
Cellular Growth ◽  
Pol I ◽  
Global Protein Synthesis ◽  
Sense And Respond ◽  
Pol Iii ◽  
Factor C

Ribosome and protein synthesis are major metabolic events that control cellular growth and proliferation. Impairment in ribosome biogenesis pathways and mRNA translation is associated with pathologies such as cancer and developmental disorders. Processes that control global protein synthesis are tightly regulated at different levels by numerous factors and linked with multiple cellular signaling pathways. Several of these merge on the growth promoting factor c-Myc, which induces ribosome biogenesis by stimulating Pol I, Pol II, and Pol III transcription. However, how cells sense and respond to mRNA translation stress is not well understood. It was more recently shown that mRNA translation stress activates c-Myc, through a specific induction of E2F1 synthesis via a PI3Kδ-dependent pathway. This review focuses on how this novel feedback pathway stimulates cellular growth and proliferation pathways to synchronize protein synthesis with ribosome biogenesis. It also describes for the first time the oncogenic activity of the mRNA, and not the encoded protein.

scholarly journals Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma

2015 ◽  
Vol 6 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Jennifer R. Devlin ◽  
Katherine M. Hannan ◽  
Nadine Hein ◽  
Carleen Cullinane ◽  
Eric Kusnadi ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Ribosome Biogenesis ◽  
Mrna Translation
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