Abstract
The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor® 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.
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References
Langer-Safer PR, Levine M, Ward DC (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci U S A 79(14):4381–4385
Manuelidis L, Langer-Safer PR, Ward DC (1982) High-resolution mapping of satellite DNA using biotin-labeled DNA probes. J Cell Biol 95(2 Pt 1):619–625
DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243(4896):1360–1363
Brown JM, Buckle VJ (2010) Detection of nascent RNA transcripts by fluorescence in situ hybridization. Methods Mol Biol 659:33–50. https://doi.org/10.1007/978-1-60761-789-1_3
Ronander E, Bengtsson DC, Joergensen L, Jensen AT, Arnot DE (2012) Analysis of single-cell gene transcription by RNA fluorescent in situ hybridization (FISH). J Vis Exp (68):4073. https://doi.org/10.3791/4073
Wagner M, Haider S (2012) New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotechnol 23(1):96–102. https://doi.org/10.1016/j.copbio.2011.10.010
Twedt DC, Cullen J, McCord K, Janeczko S, Dudak J, Simpson K (2014) Evaluation of fluorescence in situ hybridization for the detection of bacteria in feline inflammatory liver disease. J Feline Med Surg 16(2):109–117. https://doi.org/10.1177/1098612X13498249
Gribnau J, Diderich K, Pruzina S, Calzolari R, Fraser P (2000) Intergenic transcription and developmental remodeling of chromatin subdomains in the human beta-globin locus. Mol Cell 5(2):377–386
van de Corput MP, Grosveld FG (2001) Fluorescence in situ hybridization analysis of transcript dynamics in cells. Methods 25(1):111–118. https://doi.org/10.1006/meth.2001.1220
Bridger JM, Kalla C, Wodrich H, Weitz S, King JA, Khazaie K, Krausslich HG, Lichter P (2005) Nuclear RNAs confined to a reticular compartment between chromosome territories. Exp Cell Res 302(2):180–193. https://doi.org/10.1016/j.yexcr.2004.07.038
Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A (2007) A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 8:39. https://doi.org/10.1186/1471-2164-8-39
Bonifazi E, Gullotta F, Vallo L, Iraci R, Nardone AM, Brunetti E, Botta A, Novelli G (2006) Use of RNA fluorescence in situ hybridization in the prenatal molecular diagnosis of myotonic dystrophy type I. Clin Chem 52(2):319–322. https://doi.org/10.1373/clinchem.2005.056283
Renwick N, Cekan P, Masry PA, McGeary SE, Miller JB, Hafner M, Li Z, Mihailovic A, Morozov P, Brown M, Gogakos T, Mobin MB, Snorrason EL, Feilotter HE, Zhang X, Perlis CS, Wu H, Suarez-Farinas M, Feng H, Shuda M, Moore PS, Tron VA, Chang Y, Tuschl T (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123(6):2694–2702. https://doi.org/10.1172/JCI68760
Clemson CM, Lawrence JB (1996) Multifunctional compartments in the nucleus: insights from DNA and RNA localization. J Cell Biochem 62(2):181–190. https://doi.org/10.1002/(SICI)1097-4644(199608)62:2%3C181::AID-JCB6%3E3.0.CO;2-O
Jolly C, Metz A, Govin J, Vigneron M, Turner BM, Khochbin S, Vourc'h C (2004) Stress-induced transcription of satellite III repeats. J Cell Biol 164(1):25–33. https://doi.org/10.1083/jcb.200306104
Sierra-Miranda M, Delgadillo DM, Mancio-Silva L, Vargas M, Villegas-Sepulveda N, Martinez-Calvillo S, Scherf A, Hernandez-Rivas R (2012) Two long non-coding RNAs generated from subtelomeric regions accumulate in a novel perinuclear compartment in Plasmodium falciparum. Mol Biochem Parasitol 185(1):36–47. https://doi.org/10.1016/j.molbiopara.2012.06.005
Bertozzi D, Iurlaro R, Sordet O, Marinello J, Zaffaroni N, Capranico G (2011) Characterization of novel antisense HIF-1alpha transcripts in human cancers. Cell Cycle 10(18):3189–3197
Froehler BC, Wadwani S, Terhorst T, Gerrard S (1992) Oligodeoxynucleotides containing C-5 propyne analogs of 2′-deoxyuridine and 2′-deoxycytidine. Tetrahedron Lett 33:5307–5310
Barnes TW 3rd, Turner DH (2001) Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines. J Am Chem Soc 123(18):4107–4118
Prosnyak MI, Veselovskaya SI, Myasnikov VA, Efremova EJ, Potapov VK, Limborska SA, Sverdlov ED (1994) Substitution of 2-aminoadenine and 5-methylcytosine for adenine and cytosine in hybridization probes increases the sensitivity of DNA fingerprinting. Genomics 21(3):490–494. https://doi.org/10.1006/geno.1994.1306
Xodo LE, Manzini G, Quadrifoglio F, van der Marel GA, van Boom JH (1991) Effect of 5-methylcytosine on the stability of triple-stranded DNA—a thermodynamic study. Nucleic Acids Res 19(20):5625–5631
Lebedev Y, Akopyants N, Azhikina T, Shevchenko Y, Potapov V, Stecenko D, Berg D, Sverdlov E (1996) Oligonucleotides containing 2-aminoadenine and 5-methylcytosine are more effective as primers for PCR amplification than their nonmodified counterparts. Genet Anal 13(1):15–21
He J, Seela F (2002) Propynyl groups in duplex DNA: stability of base pairs incorporating 7-substituted 8-aza-7-deazapurines or 5-substituted pyrimidines. Nucleic Acids Res 30(24):5485–5496
Kawasaki AM, Casper MD, Freier SM, Lesnik EA, Zounes MC, Cummins LL, Gonzalez C, Cook PD (1993) Uniformly modified 2′-deoxy-2′-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. J Med Chem 36(7):831–841
Silverman AP, Kool ET (2007) Oligonucleotide probes for RNA-targeted fluorescence in situ hybridization. Adv Clin Chem 43:79–115
Petersen M, Wengel J (2003) LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 21(2):74–81. https://doi.org/10.1016/S0167-7799(02)00038-0
Rahman SM, Seki S, Utsuki K, Obika S, Miyashita K, Imanishi T (2007) 2′,4′-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles. Nucleosides Nucleotides Nucleic Acids 26(10–12):1625–1628. https://doi.org/10.1080/15257770701548980
Biamonti G, Vourc'h C (2010) Nuclear stress bodies. Cold Spring Harb Perspect Biol 2(6):a000695. https://doi.org/10.1101/cshperspect.a000695
Metz A, Soret J, Vourc'h C, Tazi J, Jolly C (2004) A key role for stress-induced satellite III transcripts in the relocalization of splicing factors into nuclear stress granules. J Cell Sci 117(Pt 19):4551–4558. https://doi.org/10.1242/jcs.01329
Denegri M, Chiodi I, Corioni M, Cobianchi F, Riva S, Biamonti G (2001) Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors. Mol Biol Cell 12(11):3502–3514
Rizzi N, Denegri M, Chiodi I, Corioni M, Valgardsdottir R, Cobianchi F, Riva S, Biamonti G (2004) Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock. Mol Biol Cell 15(2):543–551. https://doi.org/10.1091/mbc.E03-07-0487
Valgardsdottir R, Chiodi I, Giordano M, Rossi A, Bazzini S, Ghigna C, Riva S, Biamonti G (2008) Transcription of satellite III non-coding RNAs is a general stress response in human cells. Nucleic Acids Res 36(2):423–434. https://doi.org/10.1093/nar/gkm1056
Heckler G, Aigueperse C, Hettal L, Thuillier Q, de Chaumont F, Dallongeville S, Behm-Ansmant I (2020) Substructure analyzer: a user-friendly workflow for rapid exploration and accurate analysis of cellular bodies in fluorescence microscopy images. J Vis Exp (161). https://doi.org/10.3791/60990
Acknowledgments
V.V. and G.H. were supported by a graduate fellowship from the french Ministère Délégué à la Recherche et aux Technologies. This work was supported by grants from the European Alternative Splicing Network of Excellence (EURASNET, FP6 life sciences, genomics and biotechnology for health) and the European Associated Laboratory (LEA) on pre-mRNA splicing created by CNRS, UL, UM1, UM2 and Max Planck Institut. A. Metz and H. Kempf are acknowledged for helpful discussions. The PTIBC platform of UMS2008 is thanked for the access to the SP2 Confocal Microscope.
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Vautrot, V., Heckler, G., Aigueperse, C., Behm-Ansmant, I. (2021). Fluorescence In Situ Hybridization of Small Non-Coding RNAs. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 2300. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1386-3_8
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DOI: https://doi.org/10.1007/978-1-0716-1386-3_8
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