Hypoxia re-programs 2?-O-Me modifications on ribosomal RNA

•Chronic hypoxia stimulates RNA Pol I activity•In hypoxia, a pool of specialized rRNA translates VEGFC IRES•Hypoxia changes 2?-O-Me modification - epitranscriptomic marks on Hypoxia is one the critical stressors encountered by various cells human body under diverse pathophysiologic conditions including cancer and has profound impacts several metabolic physiologic processes. prompts internal ribosome entry site (IRES)-mediated translation key genes, such as VEGF, that are vital for tumor progression. Here, we describe remarkably upregulates Polymerase activity. We discovered in shows different methylation pattern compared to normoxia. Heterogeneity ribosomes due diversity ribosomal protein composition been postulated generate “specialized ribosomes” differentially regulate translation. find sub-set methylated recognizes VEGF-C IRES, suggesting heterogeneity allows altered functions hypoxia. common stressor. It noticeable features solid tumors determinant progression (Gilkes et al., 2014Gilkes D.M. Semenza G.L. Wirtz D. extracellular matrix: drivers tumour metastasis.Nat. Rev. Cancer. 2014; 14: 430-439Crossref PubMed Scopus (871) Google Scholar; Nakazawa 2016Nakazawa M.S. Keith B. Simon M.C. Oxygen availability adaptations.Nat. 2016; 16: 663-673Crossref (239) Scholar). Hypoxic often poorly differentiated, highly invasive, resistant treatment (Samanta 2014Samanta Gilkes Chaturvedi P. Xiang L. Hypoxia-inducible factors required chemotherapy resistance breast stem cells.Proc. Natl. Acad. Sci. U S A. 111: E5429-E5438Crossref (362) Several evidences indicate supports (Keith Simon, 2007Keith factors, cells, cancer.Cell. 2007; 129: 465-472Abstract Full Text PDF (923) Samanta Cellular adaptation predominantly driven stabilization HIF-1? transcription factor. activates target genes profoundly modulate cellular (Semenza, 2012Semenza physiology medicine.Cell. 2012; 148: 399-408Abstract (2062) also (Braunstein 2007Braunstein S. Karpisheva K. Pola C. Goldberg J. Hochman T. Yee H. Cangiarella Arju R. Formenti S.C. Schneider R.J. A hypoxia-controlled cap-dependent cap-independent switch cancer.Mol. Cell. 28: 501-512Abstract (222) In normoxic conditions, majority mRNAs initiated following recognition 7-methylguanylate cap (m7GpppN). Cells challenged with tend show mechanistic way recognize some mRNA transcripts. represses cap-mediated promoting sequestration eIF4E, result increased binding eIF4E 4E-BP1. This causes disruption eIF4F-containing initiation complex (Gebauer, 2012Gebauer F. Versatility translational machinery during stress: changing partners keep dancing.Cell Res. 22: 1634-1636Crossref (2) van den Beucken 2006van Koritzinsky M. Wouters B.G. Translational control gene expression hypoxia.Cancer Biol. Ther. 2006; 5: 749-755Crossref (124) hypoxic vascular endothelial growth factor (VEGF-C), utilize an alternative mode engages (IRES) Morfoisse 2014Morfoisse Kuchnio Frainay Gomez-Brouchet Delisle M.B. Marzi Helfer A.C. Hantelys Pujol Guillermet-Guibert al.Hypoxia induces metastatic via HIF-1alpha-independent translation-mediated mechanism.Cell Rep. 6: 155-167Abstract (88) bypass inhibition (Holcik Sonenberg, 2005Holcik Sonenberg N. stress apoptosis.Nat. Mol. Cell 2005; 318-327Crossref (1029) from (IRES-dependent) regarded attributes adaptive response microenvironment Accounting broader explanation divergent evidence, surmise blocks most eIF4F-directed while activating other eIF4F-independent but pathways addition specific contributors, IRES (Jackson, 2013Jackson The current status vertebrate IRESs.Cold Spring Harb. Perspect. 2013; a011569Crossref (107) Young 2008Young R.M. Wang S.J. Gordan J.D. Ji X. Liebhaber S.A. Hypoxia-mediated selective site-independent mechanism.J. Chem. 2008; 283: 16309-16319Abstract (95) Ribosomes macromolecular machines involving intricate assembly proteins (rRNA), sophisticated function translating messenger (Hussmann 2017Hussmann J.A. Osadnik Gross C.A. Ribosomal architecture: constraints imposed need self-production.Curr. 2017; 27: R798-R800Abstract (1) polymerase (RNA I) only transcribes DNA (rDNA) make core components, 28S, 5.8S, 18S, ribosomes. rDNA exists tandem repeats (over 200 copies total) located acrocentric chromosomes (this corresponds 13,14, 15, 21, 22) (Weeks 2019Weeks S.E. Metge B.J. Samant R.S. nucleolus: central hub drive progression.Cell Life 2019; 76: 4511-4524Crossref (57) Nucleoli non-membranous, liquid-liquid droplet-like, intra-nuclear bodies form around nucleolar organizer regions (NORs), chromosomal actively code (Feric 2016Feric Vaidya Harmon T.S. Mitrea Zhu Richardson T.M. Kriwacki R.W. Pappu R.V. Brangwynne C.P. Coexisting liquid phases underlie subcompartments.Cell. 165: 1686-1697Abstract (916) Leslie, 2014Leslie Central command.Science. 345: 506-507Crossref (8) For homeostasis, few loci be active; this reflected fewer nucleoli. However, physiologically or proliferating reliance consequently biogenesis, evident morphologically number nucleoli (Farley-Barnes 2018Farley-Barnes K.I. McCann K.L. Ogawa L.M. Merkel Surovtseva Y.V. Baserga Diverse regulators biogenesis number.Cell 2018; 1923-1934Abstract (58) Ferreira 2020Ferreira Schneekloth Jr., J.S. Panov Hannan K.M. R.D. Targeting therapy comes age.Cells. 2020; 9: 266Crossref (69) Due its utmost importance, very tightly regulated. Multiple influence (Bywater 2012Bywater M.J. Poortinga G. Sanij E. Hein Peck Cullinane Wall Cluse Drygin Anderes al.Inhibition therapeutic strategy promote cancer-specific activation p53.Cancer 51-65Abstract (393) Peltonen 2014Peltonen Colis Liu Trivedi Moubarek Moore H.M. Bai Rudek M.A. Bieberich C.J. Laiho targeting modality destruction possesses anticancer activity.Cancer 25: 77-90Abstract (173) Despite role normal disease pathophysiologies, functional activity remain largely unexplored. Here our findings chronic dramatically increase upstream factor/upstream (UBF/UBTF). Tumor triggers angiogenic switch. vascularization through elevated proangiogenic VEGF. Angiogenesis regulated multiple checks, at level (Bergers Benjamin, 2003Bergers Benjamin L.E. Tumorigenesis switch.Nat. 2003; 3: 401-410Crossref (2858) During VEGF translated using IRES. recent past, there have indications structure (Genuth Barna, 2018Genuth N.R. Barna discovery implications regulation organismal life.Mol. 71: 364-374Abstract (181) Xue 2012Xue Specialized ribosomes: new frontier biology.Nat. 13: 355-369Crossref (448) links “functional specialization” still emerging (Dinman, 2016Dinman Pathways Brussels Lecture.J. 428: 2186-2194Crossref (74) Genuth One major challenges defining lack methodology selectively isolate subpopulation responsible Haag Dinman, 2019Haag E.S. Dinman Still searching ribosomes.Dev. 48: 744-746Abstract (6) Using IRES-based model, able reveal exist sub-groups contain marked differential patterns entrusted task recognition. Intra-tumoral notable treatment-resistant implicated important (Hein 2013Hein George A.J. therapy.Trends Med. 19: 643-654Abstract (162) impact remains unknown. To test this, used two distinct models. first model comprises T47D immortalized mammary epithelial (HMECs) grown (or normoxic) second pair syngeneic cell lines, MCF10A MCF10CA1acl.1. no detectable whereas MCF10CA1acl.1 inherently high levels (Figure S1A). analyzed quantifying short-lived 5? external transcribed spacer (5?ETS) (Peltonen Scholar) 1A). results clearly revealed presence HIF-1?, significant activity, 5?ETS transcripts 1B). Concurrently, assess nascent synthesis, which species, were pulsed 5-Fluorouridine (FUrd). observed ~2 fold fluorescence intensity cultured confirming (Figures 1C, S1C, S1D). Consistent introduction stable HIF-1?; [with mutations (Pro-402/Pro-564 replaced Ala) sites E3 ubiquitin ligase VHL], into caused S1A S1B). Furthermore, silencing induced decrease evidenced FUrd incorporation S1A, S1B, S1E) multi-subunit enzyme. RPA194 catalytic largest subunit complex. controlled series molecular events ensure precise rDNA. UBTF/UBF plays prominent recruiting elongation (Potapova Gerton, 2019Potapova T.A. Gerton J.L. nucleolus context genome organization.Chromosome 109-127Crossref (31) Russell Zomerdijk, 2005Russell Zomerdijk J.C. RNA-polymerase-I-directed transcription, life works.Trends Biochem. 30: 87-96Abstract (228) Thus, will UBF well loci. ChIP primers (positions designated Figure 1A), occupancy along 1D S1F). agreement observation, transfected showed significantly S1G). Increased rates supported open chromatin structure. Histone markers H3K4Me3, H3K9ac, H3K27ac indicative (Yu 2015Yu Shen Fan Yu Z. Analysis histone modifications liver cell.Sci. 2015; 18100Crossref (19) ChIP-qPCR, overall these 1E). requisite upregulated biogenesis. Supporting notion, when lines per nucleus 1F). interested determining if seen region tumors. injected spontaneous bearing MMTV-neu mice pimonidazole mapped intra-tumoral immunohistochemical detection adducts (Ragnum 2015Ragnum H.B. Vlatkovic Lie A.K. Axcrona Julin C.H. Frikstad Hole K.H. Seierstad Lyng marker reflects transcriptional programme associated aggressive prostate cancer.Br. 112: 382-390Crossref (104) Simultaneously, consecutive sections stained AgNOR staining. Upon digital overlay sections, 1G). Thus overall, robust synthesis stress. reconfigures controls (van 2011van Magagnin M.G. Jutten Seigneuric Lambin contributor expression.Radiother. Oncol. 2011; 99: 379-384Abstract (32) Under eukaryotic translations upon Additionally, involves synergistic action 3? poly(A) tail mRNAs. certain physiological situations end-independent occurs compromises utilizes IRESes, determinants characterized active (Arcondeguy 2013Arcondeguy Lacazette Millevoi Prats Touriol VEGF-A processing, stability translation: paradigm post-transcriptional level.Nucleic Acids 41: 7997-8010Crossref (167) Bornes 2007Bornes Prado-Lourenco Bastide Zanibellato Iacovoni induction elements early ischemic stress.Circ. 100: 305-308Crossref (56) bicistronic reporter confirm activation. system, single message, renilla luciferase mechanism drives firefly luciferase. IRES-driven registered ratio (Morfoisse stabilized IRES-initiated 2A); suppressed IRES-dependent S2A). VEGF-A, described independent element influenced (Hantelys 2019Hantelys Godet David Tatin Renaud-Gabardos Diallo L.H. Ader I. Ligat Henras al.Vasohibin1, mouse cardiomyocyte trans-acting regulates hypoxia.Elife. 8: e50094Crossref (10) Scholar, 2015Morfoisse Renaud Garmy-Susini Role lymphangiogenesis.Mol. 2: e1024821Crossref (38) utilized confirmed S2B). respective activation, both S2C S2D). hypothesized would provide undergo recognizing Ribose 2?-O-methylation (2?-O-Me) abundant secondary essential accurate efficient (Erales 2017Erales Marchand V. Panthu Gillot Belin Ghayad Garcia Laforets Marcel Baudin-Baillieu al.Evidence 2'-O-methylation plasticity: intrinsic capabilities ribosomes.Proc. 114: 12934-12939Crossref (133) Krogh 2016Krogh Jansson M.D. Hafner Tehler Birkedal U. Christensen-Dalsgaard Lund A.H. Nielsen Profiling 2'-O-Me reveals subset fractionally modified positions provides evidence heterogeneity.Nucleic 44: 7884-7895Crossref (145) hypothesis, affinity tool. employed biotin-labeled bona fide sequence UTR pulldown specifically bind sequence, absence site-specific RT-qPCR-based approach 14 functionally domains isolated enriched (Belin 2009Belin Beghin Solano-Gonzalez Bezin Brunet-Manquat Textoris Mertani H.C. Dumontet Diaz J.J. Dysregulation capacity cells.PLoS One. 2009; 4: e7147Crossref (163) 2013Marcel Therizols Morel A.P. Vendrell Hacot Albaret al.p53 acts safeguard regulating fibrillarin cancer.Cancer 24: 318-330Abstract (187) (schematic 2B). These distributed 18S 28S rRNAs localized within rRNAs, i.e., decoding center (DC) rRNA, peptidyl transferase center, helix 69 (H69) (Marcel extent was determined ratio, explained Transparent methods section. note total normoxia sites. consistent both, MCF10 models, seven out fourteen 2CI 2CII). then IRES-bound noticed 2DI 2DII). implies separate Most importantly, discrete 2EI 2EII). Essentially observations change. VEGFC-IRES allowed us population had characteristic pattern. further importance modulating silenced 2?-O-methyltransferase, S2E S2F). Fibrillarin 48 hr incubated prior assaying recorded convincing 2F). considerably decreased despite Overall, data sub-pool constitutes ultimately influences initiation. exciting Moving forward, sought unravel clues underlying mechanisms group non-coding residing nucleolus, called small RNAs (snoRNA), play guiding