Abstract
Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.
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References
Rabani M, Levin JZ, Fan L et al (2011) Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 29(5):436–442
Sharova LV, Sharov AA, Nedorezov T et al (2009) Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA Res 16(1):45–58
Chen CY, Ezzeddine N, Shyu AB (2008) Messenger RNA half-life measurements in mammalian cells. Methods Enzymol 448:335–357
Ayupe AC, Tahira AC, Camargo L et al (2015) Global analysis of biogenesis, stability and sub-cellular localization of lncRNAs mapping to intragenic regions of the human genome. RNA Biol 12(8):877–892
Clark MB, Johnston RL, Inostroza-Ponta M et al (2012) Genome-wide analysis of long noncoding RNA stability. Genome Res 22(5):885–898
Dolken L, Ruzsics Z, Radle B et al (2008) High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14(9):1959–1972
Friedel CC, Dolken L, Ruzsics Z et al (2009) Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Nucleic Acids Res 37(17):e115
Ideue T, Adachi S, Naganuma T et al (2012) U7 small nuclear ribonucleoprotein represses histone gene transcription in cell cycle-arrested cells. Proc Natl Acad Sci U S A 109(15):5693–5698
Redrup L, Branco MR, Perdeaux ER et al (2009) The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development 136(4):525–530, dev.031328 [pii]
Schwanhausser B, Busse D, Li N et al (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342. doi:10.1038/nature10098
Tani H, Mizutani R, Salam KA et al (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res 22(5):947–956
Beckedorff FC, Ayupe AC, Crocci-Souza R et al (2013) The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation. PLoS Genet 9(8):e1003705
DeOcesano-Pereira C, Amaral MS, Parreira KS et al (2014) Long noncoding RNA INXS is a critical mediator of BCL-XS induced apoptosis. Nucleic Acids Res 42(13):8343–8355
Tani H, Akimitsu N (2012) Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol 9(10):1233–1238. doi:10.4161/rna.22036
Bensaude O (2011) Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription 2(3):103–108
Brueckner F, Cramer P (2008) Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation. Nat Struct Mol Biol 15(8):811–818
Kaplan CD, Larsson KM, Kornberg RD (2008) The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. Mol Cell 30(5):547–556
Weinmann R, Raskas HJ, Roeder RG (1974) Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection. Proc Natl Acad Sci U S A 71(9):3426–3439
Raha D, Wang Z, Moqtaderi Z et al (2010) Close association of RNA polymerase II and many transcription factors with Pol III genes. Proc Natl Acad Sci U S A 107(8):3639–3644
Nechaev S, Adelman K (2011) Pol II waiting in the starting gates: regulating the transition from transcription initiation into productive elongation. Biochim Biophys Acta 1809(1):34–45
Burger K, Muhl B, Harasim T et al (2012) Chemotherapeutic drugs inhibit ribosome biogenesis at various levels. J Biol Chem 285(16):12416–12425
Lam LT, Pickeral OK, Peng AC et al (2001) Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biol 2(10):RESEARCH0041
Raghavan A, Ogilvie RL, Reilly C et al (2002) Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res 30(24):5529–5538
Yang E, Van Nimwegen E, Zavolan M et al (2003) Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes. Genome Res 13(8):1863–1872, 13/8/1863 [pii]
Maekawa S, Imamachi N, Irie T et al (2015) Analysis of RNA decay factor mediated RNA stability contributions on RNA abundance. BMC Genomics 16:154
St Laurent G, Wahlestedt C, Kapranov P (2015) The Landscape of long noncoding RNA classification. Trends Genet 31(5):239–251
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Simon RM, Dobbin K (2003) Experimental design of DNA microarray experiments. Biotechniques Suppl:16–21
Munchel SE, Shultzaberger RK, Takizawa N, Weis K (2011) Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay. Mol Biol Cell 22(15):2787–2795
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Ayupe, A.C., Reis, E.M. (2017). Evaluating the Stability of mRNAs and Noncoding RNAs. In: Ørom, U. (eds) Enhancer RNAs. Methods in Molecular Biology, vol 1468. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4035-6_11
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DOI: https://doi.org/10.1007/978-1-4939-4035-6_11
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