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RNA Tagging pp 121–144Cite as

New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.

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References

  1. Tutucci E, Livingston NM, Singer RH, Wu B (2018) Imaging mRNA in vivo, from birth to death. Annu Rev Biophys 47:85–106. https://doi.org/10.1146/annurev-biophys-070317-033037

    Article  CAS  PubMed  Google Scholar 

  2. Vera M, Biswas J, Senecal A, Singer RH, Park HY (2016) Single-cell and single-molecule analysis of gene expression regulation. Annu Rev Genet 50:267–291. https://doi.org/10.1146/annurev-genet-120215-034854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135(2):216–226. https://doi.org/10.1016/j.cell.2008.09.050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Liu Y, Beyer A, Aebersold R (2016) On the dependency of cellular protein levels on mRNA abundance. Cell 165(3):535–550. https://doi.org/10.1016/j.cell.2016.03.014

    Article  CAS  PubMed  Google Scholar 

  5. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590

    Article  CAS  PubMed  Google Scholar 

  6. Femino AM, Fogarty K, Lifshitz LM, Carrington W, Singer RH (2003) Visualization of single molecules of mRNA in situ. Methods Enzymol 361:245–304

    Article  CAS  PubMed  Google Scholar 

  7. Gandhi SJ, Zenklusen D, Lionnet T, Singer RH (2011) Transcription of functionally related constitutive genes is not coordinated. Nat Struct Mol Biol 18(1):27–34. https://doi.org/10.1038/nsmb.1934

    Article  CAS  PubMed  Google Scholar 

  8. Zenklusen D, Larson DR, Singer RH (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15(12):1263–1271. https://doi.org/10.1038/nsmb.1514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Senecal A, Munsky B, Proux F, Ly N, Braye FE, Zimmer C, Mueller F, Darzacq X (2014) Transcription factors modulate c-Fos transcriptional bursts. Cell Rep 8(1):75–83. https://doi.org/10.1016/j.celrep.2014.05.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yang L, Titlow J, Ennis D, Smith C, Mitchell J, Young FL, Waddell S, Ish-Horowicz D, Davis I (2017) Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains. Methods 126:166–176. https://doi.org/10.1016/j.ymeth.2017.06.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Buxbaum AR, Wu B, Singer RH (2014) Single beta-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343:419–422. https://doi.org/10.1126/science.1242939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Duncan S, Olsson TSG, Hartley M, Dean C, Rosa S (2016) A method for detecting single mRNA molecules in Arabidopsis thaliana. Plant Methods 12:13. https://doi.org/10.1186/s13007-016-0114-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Moor AE, Golan M, Massasa EE, Lemze D, Weizman T, Shenhav R, Baydatch S, Mizrahi O, Winkler R, Golani O, Stern-Ginossar N, Itzkovitz S (2017) Global mRNA polarization regulates translation efficiency in the intestinal epithelium. Science 357:1299–1303. https://doi.org/10.1126/science.aan2399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Moffitt JR, Hao J, Bambah-Mukku D, Lu T, Dulac C, Zhuang X (2016) High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc Natl Acad Sci U S A 113(50):14456–14461. https://doi.org/10.1073/pnas.1617699113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Long X, Colonell J, Wong AM, Singer RH, Lionnet T (2017) Quantitative mRNA imaging throughout the entire Drosophila brain. Nat Methods 14(7):703–706. https://doi.org/10.1038/nmeth.4309

    Article  CAS  PubMed  Google Scholar 

  17. Battich N, Stoeger T, Pelkmans L (2013) Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat Methods 10(11):1127–1133. https://doi.org/10.1038/nmeth.2657

    Article  CAS  PubMed  Google Scholar 

  18. Choi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA (2010) Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol 28(11):1208–1212. https://doi.org/10.1038/nbt.1692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tsanov N, Samacoits A, Chouaib R, Traboulsi AM, Gostan T, Weber C, Zimmer C, Zibara K, Walter T, Peter M, Bertrand E, Mueller F (2016) smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res 44(22):e165. https://doi.org/10.1093/nar/gkw784

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Moffitt JR, Hao J, Wang G, Chen KH, Babcock HP, Zhuang X (2016) High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization. Proc Natl Acad Sci U S A 113(39):11046–11051. https://doi.org/10.1073/pnas.1612826113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lubeck E, Coskun AF, Zhiyentayev T, Ahmad M, Cai L (2014) Single-cell in situ RNA profiling by sequential hybridization. Nat Methods 11(4):360–361. https://doi.org/10.1038/nmeth.2892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shah S, Lubeck E, Zhou W, Cai L (2017) seqFISH accurately detects transcripts in single cells and reveals robust spatial organization in the hippocampus. Neuron 94(4):752–758. https://doi.org/10.1016/j.neuron.2017.05.008

    Article  CAS  PubMed  Google Scholar 

  23. Eng CL, Lawson M, Zhu Q, Dries R, Koulena N, Takei Y, Yun J, Cronin C, Karp C, Yuan GC, Cai L (2019) Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568:235–239. https://doi.org/10.1038/s41586-019-1049-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shah S, Takei Y, Zhou W, Lubeck E, Yun J, Eng CL, Koulena N, Cronin C, Karp C, Liaw EJ, Amin M, Cai L (2018) Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH. Cell 174(2):363–376. https://doi.org/10.1016/j.cell.2018.05.035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bayer LV, Batish M, Formel SK, Bratu DP (2015) Single-molecule RNA in situ hybridization (smFISH) and immunofluorescence (IF) in the Drosophila egg chamber. Methods Mol Biol 1328:125–136. https://doi.org/10.1007/978-1-4939-2851-4_9

    Article  CAS  PubMed  Google Scholar 

  26. Eliscovich C, Shenoy SM, Singer RH (2017) Imaging mRNA and protein interactions within neurons. Proc Natl Acad Sci U S A 114(10):E1875–E1884. https://doi.org/10.1073/pnas.1621440114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Buxbaum AR, Haimovich G, Singer RH (2015) In the right place at the right time: visualizing and understanding mRNA localization. Nat Rev Mol Cell Biol 16(2):95–109. https://doi.org/10.1038/nrm3918

    Article  CAS  PubMed  Google Scholar 

  28. Weil TT, Parton RM, Davis I (2010) Making the message clear: visualizing mRNA localization. Trends Cell Biol 20(7):380–390. https://doi.org/10.1016/j.tcb.2010.03.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136(4):719–730. https://doi.org/10.1016/j.cell.2009.01.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lecuyer E, Yoshida H, Parthasarathy N, Alm C, Babak T, Cerovina T, Hughes TR, Tomancak P, Krause HM (2007) Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131(1):174–187. https://doi.org/10.1016/j.cell.2007.08.003

    Article  CAS  PubMed  Google Scholar 

  31. Samacoits A, Chouaib R, Safieddine A, Traboulsi AM, Ouyang W, Zimmer C, Peter M, Bertrand E, Walter T, Mueller F (2018) A computational framework to study sub-cellular RNA localization. Nat Commun 9(1):4584. https://doi.org/10.1038/s41467-018-06868-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Das S, Singer RH, Yoon YJ (2019) The travels of mRNAs in neurons: do they know where they are going? Curr Opin Neurobiol 57:110–116. https://doi.org/10.1016/j.conb.2019.01.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bahar Halpern K, Tanami S, Landen S, Chapal M, Szlak L, Hutzler A, Nizhberg A, Itzkovitz S (2015) Bursty gene expression in the intact mammalian liver. Mol Cell 58(1):147–156. https://doi.org/10.1016/j.molcel.2015.01.027

    Article  CAS  PubMed  Google Scholar 

  34. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4(10):e309. https://doi.org/10.1371/journal.pbio.0040309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Paul B, Montpetit B (2016) Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus. Mol Biol Cell 27(17):2742–2756. https://doi.org/10.1091/mbc.E16-04-0244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bahar Halpern K, Caspi I, Lemze D, Levy M, Landen S, Elinav E, Ulitsky I, Itzkovitz S (2015) Nuclear retention of mRNA in mammalian tissues. Cell Rep 13(12):2653–2662. https://doi.org/10.1016/j.celrep.2015.11.036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Trcek T, Larson DR, Moldon A, Query CC, Singer RH (2011) Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147(7):1484–1497. https://doi.org/10.1016/j.cell.2011.11.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Trcek T, Sato H, Singer RH, Maquat LE (2013) Temporal and spatial characterization of nonsense-mediated mRNA decay. Genes Dev 27(5):541–551. https://doi.org/10.1101/gad.209635.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pichon X, Lagha M, Mueller F, Bertrand E (2018) A growing toolbox to image gene expression in single cells: sensitive approaches for demanding challenges. Mol Cell 71(3):468–480. https://doi.org/10.1016/j.molcel.2018.07.022

    Article  CAS  PubMed  Google Scholar 

  40. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445

    Article  CAS  PubMed  Google Scholar 

  41. Peabody DS (1993) The RNA binding site of bacteriophage MS2 coat protein. EMBO J 12(2):595–600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fusco D, Bertrand E, Singer RH (2004) Imaging of single mRNAs in the cytoplasm of living cells. Prog Mol Subcell Biol 35:135–150

    Article  PubMed  PubMed Central  Google Scholar 

  43. Tutucci E, Vera M, Singer RH (2018) Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system. Nat Protoc 13(10):2268–2296. https://doi.org/10.1038/s41596-018-0037-2

    Article  CAS  PubMed  Google Scholar 

  44. Brody Y, Neufeld N, Bieberstein N, Causse SZ, Bohnlein EM, Neugebauer KM, Darzacq X, Shav-Tal Y (2011) The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol 9(1):e1000573. https://doi.org/10.1371/journal.pbio.1000573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy SM, Phair RD, Singer RH (2007) In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol 14(9):796–806. https://doi.org/10.1038/nsmb1280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Schmidt U, Basyuk E, Robert MC, Yoshida M, Villemin JP, Auboeuf D, Aitken S, Bertrand E (2011) Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation. J. Cell Biol 193(5):819–829. https://doi.org/10.1083/jcb.201009012

    Article  CAS  Google Scholar 

  47. Grunwald D, Singer RH (2010) In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport. Nature 467(7315):604–607. https://doi.org/10.1038/nature09438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Smith C, Lari A, Derrer CP, Ouwehand A, Rossouw A, Huisman M, Dange T, Hopman M, Joseph A, Zenklusen D, Weis K, Grunwald D, Montpetit B (2015) In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p. J Cell Biol 211(6):1121–1130. https://doi.org/10.1083/jcb.201503135

    Article  PubMed  PubMed Central  Google Scholar 

  49. Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8(2):165–170. https://doi.org/10.1038/nmeth.1551

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH (2014) Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343(6169):422–424. https://doi.org/10.1126/science.1239200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Halstead JM, Lionnet T, Wilbertz JH, Wippich F, Ephrussi A, Singer RH, Chao JA (2015) Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science 347(6228):1367–1671. https://doi.org/10.1126/science.aaa3380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Katz ZB, English BP, Lionnet T, Yoon YJ, Monnier N, Ovryn B, Bathe M, Singer RH (2016) Mapping translation ‘hot-spots’ in live cells by tracking single molecules of mRNA and ribosomes. elife 5:e10415

    Article  PubMed  PubMed Central  Google Scholar 

  53. Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL, Lionnet T, Stasevich TJ (2016) Real-time quantification of single RNA translation dynamics in living cells. Science 352(6292):1425–1429. https://doi.org/10.1126/science.aaf0899

    Article  CAS  PubMed  Google Scholar 

  54. Pichon X, Bastide A, Safieddine A, Chouaib R, Samacoits A, Basyuk E, Peter M, Mueller F, Bertrand E (2016) Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells. J Cell Biol 214(6):769–781. https://doi.org/10.1083/jcb.201605024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wu B, Eliscovich C, Yoon YJ, Singer RH (2016) Translation dynamics of single mRNAs in live cells and neurons. Science 352(6292):1430–1435. https://doi.org/10.1126/science.aaf1084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Garcia JF, Parker R (2015) MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21(8):1393–1395. https://doi.org/10.1261/rna.051797.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Haimovich G, Zabezhinsky D, Haas B, Slobodin B, Purushothaman P, Fan L, Levin JZ, Nusbaum C, Gerst JE (2016) Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to “MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system”. RNA 22(5):660–666. https://doi.org/10.1261/rna.055095.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Tutucci E, Vera M, Biswas J, Garcia J, Parker R, Singer RH (2018) An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15(1):81–89. https://doi.org/10.1038/nmeth.4502

    Article  CAS  PubMed  Google Scholar 

  59. Heinrich S, Sidler CL, Azzalin CM, Weis K (2017) Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. RNA 23(2):134–141. https://doi.org/10.1261/rna.057786.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Tantale K, Mueller F, Kozulic-Pirher A, Lesne A, Victor JM, Robert MC, Capozi S, Chouaib R, Backer V, Mateos-Langerak J, Darzacq X, Zimmer C, Basyuk E, Bertrand E (2016) A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat Commun 7:12248. https://doi.org/10.1038/ncomms12248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Chao JA, Patskovsky Y, Almo SC, Singer RH (2008) Structural basis for the coevolution of a viral RNA-protein complex. Nat Struct Mol Biol 15(1):103–105. https://doi.org/10.1038/nsmb1327

    Article  CAS  PubMed  Google Scholar 

  62. Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332(6028):475–478. https://doi.org/10.1126/science.1202142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Saroufim MA, Bensidoun P, Raymond P, Rahman S, Krause MR, Oeffinger M, Zenklusen D (2015) The nuclear basket mediates perinuclear mRNA scanning in budding yeast. J Cell Biol 211(6):1131–1140. https://doi.org/10.1083/jcb.201503070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wang C, Han B, Zhou R, Zhuang X (2016) Real-time imaging of translation on single mrna transcripts in live cells. Cell 165(4):990–1001. https://doi.org/10.1016/j.cell.2016.04.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Yan X, Hoek TA, Vale RD, Tanenbaum ME (2016) Dynamics of translation of single mRNA molecules In vivo. Cell 165(4):976–989. https://doi.org/10.1016/j.cell.2016.04.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Das S, Moon HC, Singer RH, Park HY (2018) A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons. Sci Adv 4(6):eaar3448. https://doi.org/10.1126/sciadv.aar3448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Chen J, Nikolaitchik O, Singh J, Wright A, Bencsics CE, Coffin JM, Ni N, Lockett S, Pathak VK, Hu WS (2009) High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis. Proc Natl Acad Sci U S A 106(32):13535–13540. https://doi.org/10.1073/pnas.0906822106

    Article  PubMed  PubMed Central  Google Scholar 

  68. Lange S, Katayama Y, Schmid M, Burkacky O, Brauchle C, Lamb DC, Jansen RP (2008) Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9(8):1256–1267. https://doi.org/10.1111/j.1600-0854.2008.00763.x

    Article  CAS  PubMed  Google Scholar 

  69. Brodsky AS, Silver PA (2002) Identifying proteins that affect mRNA localization in living cells. Methods 26(2):151–155. https://doi.org/10.1016/S1046-2023(02)00017-8

    Article  CAS  PubMed  Google Scholar 

  70. Takizawa PA, Vale RD (2000) The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc Natl Acad Sci U S A 97(10):5273–5278. https://doi.org/10.1073/pnas.080585897

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, Shu X, Zhang J, Tsien RY (2017) The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem Sci 42(2):111–129. https://doi.org/10.1016/j.tibs.2016.09.010

    Article  CAS  PubMed  Google Scholar 

  72. Specht EA, Braselmann E, Palmer AE (2017) A critical and comparative review of fluorescent tools for live-cell imaging. Annu Rev Physiol 79:93–117. https://doi.org/10.1146/annurev-physiol-022516-034055

    Article  CAS  PubMed  Google Scholar 

  73. Cranfill PJ, Sell BR, Baird MA, Allen JR, Lavagnino Z, de Gruiter HM, Kremers GJ, Davidson MW, Ustione A, Piston DW (2016) Quantitative assessment of fluorescent proteins. Nat Methods 13(7):557–562. https://doi.org/10.1038/nmeth.3891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Scott DJ, Gunn NJ, Yong KJ, Wimmer VC, Veldhuis NA, Challis LM, Haidar M, Petrou S, Bathgate RAD, Griffin MDW (2018) A novel ultra-stable, monomeric green fluorescent protein for direct volumetric imaging of whole organs using CLARITY. Sci Rep 8(1):667. https://doi.org/10.1038/s41598-017-18045-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Feng S, Sekine S, Pessino V, Li H, Leonetti MD, Huang B (2017) Improved split fluorescent proteins for endogenous protein labeling. Nat Commun 8(1):370. https://doi.org/10.1038/s41467-017-00494-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Slubowski CJ, Funk AD, Roesner JM, Paulissen SM, Huang LS (2015) Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy. Yeast 32(4):379–387. https://doi.org/10.1002/yea.3065

    Article  CAS  PubMed  Google Scholar 

  77. Lim F, Peabody DS (1994) Mutations that increase the affinity of a translational repressor for RNA. Nucleic Acids Res 22(18):3748–3752

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33–38

    Article  CAS  PubMed  Google Scholar 

  79. Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569):913–916. https://doi.org/10.1126/science.1068539

    Article  CAS  PubMed  Google Scholar 

  80. Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24(1):79–88. https://doi.org/10.1038/nbt1172

    Article  CAS  PubMed  Google Scholar 

  81. Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5):407–409. https://doi.org/10.1038/nmeth.2413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Mueller F, Senecal A, Tantale K, Marie-Nelly H, Ly N, Collin O, Basyuk E, Bertrand E, Darzacq X, Zimmer C (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4):277–278. https://doi.org/10.1038/nmeth.2406

    Article  CAS  PubMed  Google Scholar 

  83. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7(10):R100. https://doi.org/10.1186/gb-2006-7-10-r100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by NIH Grant GM57071 to R.H.S., and by an ANRS Grant to E.B. E.T. was supported by Swiss National Science Foundation Fellowships P2GEP3_155692 and P300PA_164717. X.P. was supported by a fellowship from the Labex EpiGenMed Montpellier/Université de Montpellier, and E.B. had a travel grant from the Philippe Foundation.

Contributions: X.P. performed the experiments and wrote the protocol for mammalian cells. M.C.R. performed the experiments testing different FP in yeast. E.T. performed experiments and designed the protocol for yeast. E.T. wrote the manuscript with inputs from E.B. and R.H.S.

Author information

Author notes

  1. Evelina Tutucci

    Present address: Systems Biology Lab, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Authors and Affiliations

  1. Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Montpellier, France

    Xavier Pichon, Marie-Cécile Robert & Edouard Bertrand

  2. Equipe labélisée Ligue Nationale Contre le Cancer, Montpellier, France

    Xavier Pichon

  3. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Edouard Bertrand, Robert H. Singer & Evelina Tutucci

  4. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA

    Robert H. Singer

  5. Janelia Research Campus of the HHMI, Ashburn, VA, USA

    Robert H. Singer

Authors
  1. Xavier Pichon
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  2. Marie-Cécile Robert
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  3. Edouard Bertrand
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  4. Robert H. Singer
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  5. Evelina Tutucci
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Corresponding author

Correspondence to Evelina Tutucci .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

1 Electronic Supplementary Material

Supplementary Video 1

MDN1 mRNAs tagged with 24xMBSV6 in the 3′UTR and labeled with MCP-NLS-2xyeGFP are shown in gray; Nup49-tdTomato is shown in red. One single Z-plane was streamed at 100 ms intervals for a total of 100 frames. The white arrowhead indicates a transcription site in the nucleus, brighter than single spots in the cytoplasm. The blue arrowheads indicate single mRNAs in the cytoplasm. Scale bar 3 μm (MP4 136 kb)

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Pichon, X., Robert, MC., Bertrand, E., Singer, R.H., Tutucci, E. (2020). New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

  • eBook Packages: Springer Protocols

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