Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing DOI Creative Commons

Yana Rozevsky,

Tal Gilboa, Xander F. van Kooten

et al.

ACS Nano, Journal Year: 2020, Volume and Issue: 14(10), P. 13964 - 13974

Published: Sept. 15, 2020

RNA quantification methods are broadly used in life science research and clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for quantification. However, cases of rare transcripts or inhibiting contaminants sample, an extensive amplification could bias copy number estimation, leading to errors false diagnosis. Single-molecule techniques may bypass but commonly rely on fluorescence detection probe hybridization, which introduces noise limits multiplexing. Here, we introduce quantitative nanopore sensing (RT-qNP), method that involves synthesis single-molecule gene-specific cDNAs without need purification amplification. RT-qNP allows us accurately quantify relative expression metastasis-associated genes MACC1 S100A4 nonmetastasizing metastasizing human cell lines, even at levels RT-qPCR produces uncertain results. We further demonstrate versatility by adapting it severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) against a reference gene. This internal circumvents producing calibration curve each measurement, imminent requirement experiments. In summary, describe general process complicated biological samples with minimal losses, adequate direct sensing. Thus, harnessing sensitivity label-free counting, can potentially detect minute biomarkers viral infection early stages disease provide accurate amplification-free

Language: Английский

RNA sequencing: the teenage years DOI
Rory Stark, Marta Grzelak, James Hadfield

et al.

Nature Reviews Genetics, Journal Year: 2019, Volume and Issue: 20(11), P. 631 - 656

Published: July 24, 2019

Language: Английский

Citations

1566
High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue DOI Creative Commons
Yang Liu, Mingyu Yang, Yanxiang Deng

et al.

Cell, Journal Year: 2020, Volume and Issue: 183(6), P. 1665 - 1681.e18

Published: Nov. 13, 2020

Language: Английский

Citations

660
A Single-Cell RNA Sequencing Profiles the Developmental Landscape of Arabidopsis Root DOI Creative Commons
Tian‐Qi Zhang, Zhou-Geng Xu, Guan-Dong Shang

et al.

Molecular Plant, Journal Year: 2019, Volume and Issue: 12(5), P. 648 - 660

Published: April 17, 2019

Language: Английский

Citations

406
Intracellular mRNA transport and localized translation DOI
Sulagna Das, Mariá Vera, Valentina Gandin

et al.

Nature Reviews Molecular Cell Biology, Journal Year: 2021, Volume and Issue: 22(7), P. 483 - 504

Published: April 9, 2021

Language: Английский

Citations

272
A single-cell analysis of the Arabidopsis vegetative shoot apex DOI Creative Commons
Tian‐Qi Zhang, Yu Chen, Jiawei Wang

et al.

Developmental Cell, Journal Year: 2021, Volume and Issue: 56(7), P. 1056 - 1074.e8

Published: March 18, 2021

Language: Английский

Citations

226
Filamentous phages: masters of a microbial sharing economy DOI Creative Commons
Iain D. Hay, Trevor Lithgow

EMBO Reports, Journal Year: 2019, Volume and Issue: 20(6)

Published: April 5, 2019

Language: Английский

Citations

167
Mechanisms tailoring the expression of heat shock proteins to proteostasis challenges DOI Creative Commons
Lokha R. Alagar Boopathy, Suleima Jacob‐Tomas, Célia Alecki

et al.

Journal of Biological Chemistry, Journal Year: 2022, Volume and Issue: 298(5), P. 101796 - 101796

Published: March 3, 2022

All cells possess an internal stress response to cope with environmental and pathophysiological challenges. Upon stress, reprogram their molecular functions activate a survival mechanism known as the heat shock response, which mediates rapid induction of chaperones such proteins (HSPs). This potent production overcomes general suppression gene expression results in high levels HSPs subsequently refold or degrade misfolded proteins. Once damage is repaired removed, terminate resume regular functions. Thus, fulfillment requires swift robust coordination between activation completion that determined by status cell. In recent years, single-cell fluorescence microscopy techniques have begun be used unravelling HSP-gene pathways, from DNA transcription mRNA degradation. this review, we will address mechanisms different organisms cell types coordinate signaling networks act transcription, translation, decay ensure protein quality control. For grow function properly, they must maintain specific cellular conditions allow acquire functional conformations achieve homeostasis (proteostasis) (1Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Genomic programs yeast changes.Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Google Scholar). Maintaining proteostasis becomes critical when facing abrupt changes external conditions, increase temperature, can lead misfolding aggregation, consequently, dysfunction (2van Oosten-Hawle P. Morimoto R.I. Organismal proteostasis: Role cell-nonautonomous regulation transcellular chaperone signaling.Genes Dev. 2014; 28: 1533-1543Crossref Scopus (60) sense, rapidly respond, adapt new for survival. Organisms bacteria mammals evolved similar varying responses successfully. Some these strategies include modulations cascades, transcriptional programs, posttranslational modifications, dynamic assembly RNA condensates (ribonucleoprotein [RNP] granules) through liquid–liquid phase separation Scholar, 3de la Fuente M. Valera S. Martínez-Guitarte J.L. ncRNAs thermoregulation: A view prokaryotes eukaryotes.FEBS Lett. 2012; 586: 4061-4069Crossref (0) 4Protter D.S.W. Parker R. Principles properties granules.Trends Cell 2016; 26: 668-679Abstract Full Text PDF (621) 5Pohl C. Dikic I. Cellular control ubiquitin-proteasome system autophagy.Science. 2019; 366: 818-822Crossref (267) 6Pomatto L.C.D. Davies K.J.A. The role declining adaptive ageing.J. Physiol. 2017; 595: 7275-7309Crossref (76) 7Yasuda Tsuchiya H. Kaiho A. Guo Q. Ikeuchi K. Endo Arai N. Ohtake F. Murata Inada T. Baumeister W. Fernández-Busnadiego Tanaka Saeki Y. Stress- ubiquitylation-dependent proteasome.Nature. 2020; 578: 296-300Crossref (84) Several converge sustain sudden acute conditions. Increases temperature universal challenge encountered most organisms. historical reasons, thermal has been paradigm study response. Nowadays, studies additional relevance due increased exposure heatwaves derived climate change (8Tomanek L. importance physiological limits determining biogeographical range shifts global change: heat-shock response.Physiol. Biochem. Zool. 2008; 81: 709-717Crossref (114) 9Kassahn K.S. Crozier R.H. Pörtner H.O. Caley M.J. Animal performance stress: Responses tolerance at biological organisation.Biol. Rev. Camb. Philos. Soc. 2009; 84: 277-292Crossref (174) 10Sengupta Garrity Sensing temperature.Curr. 2013; 23: R304-R307Abstract (72) Increased energy result heat-induced denaturation thermally altered metabolic activity leading reactive oxygen species, all macromolecules, including (11Somero G.N. temperature: Function, regulation, evolution.J. Exp. Zool Ecol. Integr. 333: 379-397Crossref (44) Cells load unfolded modulating chaperones, also (HSPs) (12Parsell D.A. Lindquist tolerance: Degradation reactivation damaged proteins.Annu. Genet. 1993; 27: 437-496Crossref 13Kültz Molecular evolutionary basis OF response.Annu. 2005; 67: 225-257Crossref (977) 14Daugaard Rohde Jäättelä 70 family: Highly homologous overlapping distinct functions.FEBS 2007; 581: 3702-3710Crossref (830) 15Rosenzweig Nillegoda N.B. Mayer M.P. Bukau B. Hsp70 network.Nat. Mol. 20: 665-680Crossref (295) (HSR) refers HSPs, it common widely studied stress. play central lifecycle because promote folding nascent polypeptides into native/functional configurations prevent aggregation 16Morimoto response: Systems biology proteotoxic aging disease.Cold Spring Harb. Symp. Quant 2011; 76: 91-99Crossref (263) collaborate mechanisms, system, autophagy, target aggregates whose native state cannot recovered degradation (5Pohl 17Wang Le W.D. Autophagy system.Adv. Med. 1206: 527-550Crossref (37) Given are network, undertake several adaptations favor synthesis expense decreasing (Fig. 1). Biochemical approaches highlight unique HSP expression. spatiotemporal resolution precise now being uncovered using high-resolution quantitative microscopy. Gene adaptions during together protect macromolecules promptly cytoplasmic nuclear activities once permissive restored (3de coordinates other protective like formation RNP integrated (ISR) repress translation initiation. ISR HSR actions endoplasmic reticulum (ER) mitochondria preserve across compartments. ranging plants genes encoding HSPs. grouped families based on apparent weight (18Jayaraj G.G. Hipp M.S. Hartl F.U. Functional modules network.Cold Perspect. 12a033951Crossref (47) 19Kampinga H.H. Hageman J. Vos Kubota Tanguay R.M. Bruford E.A. Cheetham M.E. Chen Hightower L.E. Guidelines nomenclature human proteins.Cell Stress Chaperones. 14: 105-111Crossref (813) HSP70 HSP90 functionally relevant (15Rosenzweig 20Abisambra J.F. Blair L.J. Hill S.E. Jones J.R. Kraft Rogers Koren Jinwal U.K. Lawson Johnson A.G. Wilcock O'Leary J.C. Jansen-West Muschol Golde T.E. et al.Phosphorylation dynamics regulate Hsp27-mediated rescue neuronal plasticity deficits tau transgenic mice.J. Neurosci. 2010; 30: 15374-15382Crossref (78) They ATP-dependent cooperate small HSP110. Cochaperones J-domain family modulate accelerating ATP hydrolysis, participating substrate recognition refolding 2) (21Gamerdinger Hajieva Kaya A.M. Wolfrum U. Behl Protein involves recruitment macroautophagy pathway BAG3.EMBO 889-901Crossref (385) 22Kumar Ambasta R.K. Veereshwarayya V. Rosen K.M. Kosik Band Mestril Patterson Querfurth H.W. CHIP interact beta-APP proteasome-dependent manner influence Abeta metabolism.Hum. 16: 848-864Crossref 23Lindquist 1986; 55: 1151-1191Crossref 24Lindquist Craig 1988; 22: 631-677Crossref 25Liu Liang Zhou Structural analysis Hsp70/Hsp40 system.Protein Sci. 29: 378-390Crossref (28) 26Petrucelli Dickson Kehoe Taylor Snyder Grover De Lucia McGowan E. Lewis Prihar Kim Dillmann W.H. Browne Hall Voellmy al.CHIP ubiquitination, aggregation.Hum. 2004; 13: 703-714Crossref (560) 27Ritossa puffing pattern induced DNP drosophila.Experientia. 1962; 18: 571-573Crossref (1424) further categorized constitutive inducible steady-state levels. some upregulated extent upon Among them, fastest (23Lindquist Interestingly, highly conserved among species having amino acid similarity 50% Homo sapiens Escherichia coli, while domains 96% similar, highlights its vital adaption changing (28Sørensen J.G. Kristensen T.N. Loeschcke ecological proteins: Heat proteins.Ecol. 2003; 6: 1025-1037Crossref Scholar).Figure 2The HSC70/HSP70 retaining proteostasis. illustration depicts significant tasks network inside (Starting top left tile) Under nonstress HSC70 provides cotranslational polypeptide obtain conformation; helps proteins; transports cytoplasm where assisted mitochondrial (mtHSP70) HSP60 attain involved complex and/or disassembly; leads lysosome chaperone-mediated autophagy (236Massey Kiffin Cuervo Pathophysiology autophagy.Int. 36: 2420-2434Crossref (148) 237Majeski A.E. Dice Mechanisms 2435-2444Crossref (299) (Continuing bottom During lack exit ribosome tunnel represses elongation stage. resolves granules so sequestered mRNAs recovery stress; targets terminally proteasomal degradation; autophagosome. HSP, protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) frame context undertaken eukaryotic temperature. We compare mounted suggest technological overcome gap our knowledge one main Their occurs downregulation constitutively expressed genes. Most short (around 2500 nucleotides) intronless, promoter contains more binding sites, elements (HSEs), association master factor 1 (HSF1) (29Brocchieri Conway de Macario A.J. hsp70 genome: Conservation differentiation patterns predict wide array specialized functions.BMC Evol. 8: 19Crossref (178) not expressed. However, loci neither present compact heterochromatin domain nor marked repressive epigenetic histone modification. 3′ end nucleosome-free body covered nucleosomes. bound paused polymerase II (RNAPII) (30Petesch S.J. Lis J.T. Rapid, transcription-independent loss nucleosomes over large chromatin loci.Cell. 134: 74-84Abstract (250) These characteristics stable repression facilitate prompt HSF1. HSF1 shuttles nucleus cytoplasm, kept inactive monomer members families. released trimerizes, localizes binds HSE, comprised least three nGAAn repeats organized head tail promoters products (31Anckar Sistonen Regulation Implications disease.Annu. 80: 1089-1115Crossref (462) 32Vihervaara glance.J. 127: 261-266Crossref (169) Scholar) 3). domains, oligomerization next N terminus, trans-activation C terminus induces initiation elongation, regulatory middle negatively regulates By forming trimer, affinity HSE increases each trimer repeat domain. sufficient accompanied extensive modifications. undergoes hyperphosphorylation serine threonine residues cover up 90% (33Björk J.K. mammalian family.FEBS 277: 4126-4139Crossref 34Gomez-Pastor Burchfiel E.T. Thiele D.J. factors roles physiology disease.Nat. 2018; 19: 4-19Crossref (272) 35Guettouche Boellmann Lane W.S. Analysis phosphorylation experiencing stress.BMC 4Crossref (222) 36Nakai HSF regulation.Nat. Struct. 93-95Crossref (15) only few serines 230 326, necessary (35Guettouche 37Boellmann Guettouche Fenna Mnayer DAXX interacts enhances activity.Proc. Natl. Acad. 101: 4100-4105Crossref (70) Concomitantly, sumo groups inhibitory effect removed (38Hietakangas Anckar Blomster H.A. Fujimoto Palvimo J.J. Nakai PDSM, motif phosphorylation-dependent SUMO modification.Proc. 2006; 103: 45-50Crossref (374) acetylation lysines 116 118 favors activity, whereas lysine localization Acetylation hours after decrease (39Westerheide S.D. Stevens S.M. Stress-inducible deacetylase SIRT1.Science. 323: 1063-1066Crossref (530) summary, regulated under various stresses. Although modifications identified, many others, well responsible remains elucidated. combination titration demonstrated Recent work yeast, Saccharomyces cerevisiae, allowed building simple mathematical model points dissociation HSP70/HSP90 first "switch on" step feedback switch off (40Krakowiak Zheng X. Patel Feder Z.A. Anandhakumar Valerius Gross D.S. Khalil A.S. Pincus Hsf1 constitute two-component loop response.Elife. 7e31668Crossref (33) 41Zheng Krakowiak Beyzavi Ezike Dynamic phosphorylation.Elife. 5e18638Crossref (97) al. (41Zheng identified sites were able phosphorylations no but instead favoring mediator complex. Additionally, eEF1A noncoding HSR1 activating form nucleoprotein stimulate trimerization (42Shamovsky Ivannikov Kandel E.S. Gershon Nudler RNA-mediated cells.Nature. 440: 556-560Crossref (271) Following shock, recruits multiple cofactors (43Chen Yu Yang Temple Harbinski Gao Wilson Pagliarini Identification mixed lineage leukemia 1(MLL1) coactivator 1(HSF1) 90 (HSP90) inhibition.J. Chem. 289: 18914-18927Abstract 44Jonkers Getting speed II.Nat. 2015; 167-177Crossref (468) 45Mason P.B. Cooperative competitive interactions promoter.J. 1997; 272: 33227-33233Abstract 46Park J.M. Werner Y.J. Mediator, holoenzyme, directly recruited shock.Mol. 2001; 9-19Abstract (113) Scholar), SGO2, subunit MED12, essential strong (47Takii Matsumoto Srivastava Katiyar Nakayama K.I. pericentromeric shugoshin 2 cooperates Pol recruitment.EMBO 38e102566Crossref (11) SGO2 hypophosphorylated RNAPII Transcription then P-TEFb, mediated (48Lis Mason Peng Price D.H. P-TEFb kinase loci.Genes 792-803Crossref induce C-terminal RNAPII, 49Marshall N.F. Xie Z. Control potential novel Carboxyl-terminal kinase∗.J. 1996; 271: 27176-27183Abstract positioned along removed. remodelers SWI/SNF FACT Spt6 Drosophila melanogaster (D. melanogaster) within minutes remove Besides HSF1, depends relocation membrane speckles (50Khanna Hu Belmont transgene directed motion facilitates activation.Curr. 24: 1138-1144Abstract (85) 51Vera Singer regulation: jumps shocked.Curr. R396-R398Abstract rapid, active, unidirectional movement actin polymerization. locus sequence determines contain 2–phosphorylated components machinery, speckle yet identified. coalesce discrete spots stimulation. interallelic clustering interaction HSP104 HSP12 transcription. suggested presence factories formed could coregulated (52Chowdhary Kainth undergo alteration three-dimensional structure genome organization stress.Mol. 37e00292-17Crossref (17) upregulation non-HSP cytoskeleton oxidative massive thousands (For review: (53Vihervaara Duarte F.M. driving responses.Nat. 385-397Crossref (98) Scholar)). Detailed position polymerases, landscape explain preferences (54Mueller Mieczkowski Kundu Wang Sadreyev Tolstorukov M.Y. Kingston R.E. Widespread nucleosome accessibility without occupancy induction.Genes 31: 451-462Crossref (55) 55Vihervaara Mahat D.B. Guertin Chu Danko C.G. Transcriptional pre-wired enhancer architecture.Nat. Commun. 255Crossref (65) does remodeling topology associated S2 (56Ray Munn P.R. Vihervaara Ozer Chromatin conformation cha

Language: Английский

Citations

103
FISH-quant v2: a scalable and modular tool for smFISH image analysis DOI Open Access

Arthur Imbert,

Wei Ouyang, Adham Safieddine

et al.

RNA, Journal Year: 2022, Volume and Issue: 28(6), P. 786 - 795

Published: March 28, 2022

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule fluorescence situ hybridization (smFISH) widely used single-cell-single-molecule imaging technique enabling quantitative studies its regulatory mechanisms. Today, these methods are applicable at large scale, which turn come with need for adequate tools data analysis exploration. Here, we present FISH-quant v2, highly modular tool accessible both experts non-experts. Our user-friendly package allows the user to segment nuclei cells, detect isolated RNAs, decompose dense clusters, quantify patterns visualize results single-cell level variations within cell population. This was validated applied on large-scale smFISH image sets, revealing diverse subcellular surprisingly high degree cell-to-cell heterogeneity.

Language: Английский

Citations

93
Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs DOI Creative Commons
Jérémy Dufourt, Antonio Trullo,

Jennifer Hunter

et al.

Nature Communications, Journal Year: 2018, Volume and Issue: 9(1)

Published: Nov. 29, 2018

Abstract Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and the establishment of transcriptional competence. However, impact enhancer priming by pioneer on temporal control gene expression mitotic memory remains unclear. Here we employ quantitative live imaging methods mathematical modeling test effect factor Zelda dynamics in Drosophila embryos. We demonstrate that increasing number binding sites accelerates kinetics nuclei activation regardless their past. Despite its known pioneering activities, show does not remain detectably associated with chromosomes is neither necessary nor sufficient foster memory. further reveal forms sub-nuclear dynamic hubs where events are transient. propose facilitates accumulating microenvironments it could accelerate duration multiple pre-initiation steps.

Language: Английский

Citations

140
A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting DOI Creative Commons

Racha Chouaib,

Adham Safieddine, Xavier Pichon

et al.

Developmental Cell, Journal Year: 2020, Volume and Issue: 54(6), P. 773 - 791.e5

Published: Aug. 11, 2020

Language: Английский

Citations

119