Abstract
In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. piRNA pathway mutations lead to overexpression and mobilization of retrotransposons in the germline. In different organisms, small RNAs were shown to be implicated in the posttranscriptional degradation of mRNA and/or transcriptional repression of the homologous locus. In Drosophila, the mechanism of piRNA-mediated silencing is still far from being understood. Transcriptional silencing implies a piRNA-mediated formation of repressive chromatin which diminishes the transcriptional capacity of the target locus. Nuclear Run-On (NRO) assay allows a direct estimation of the density of transcribing polymerases at specific genomic regions. Here we describe the NRO protocol on Drosophila ovarian tissues which can be useful for investigation of the transcriptional silencing in the female germline.
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References
Aravin AA, Hannon GJ, Brennecke J (2007) The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318:761–764
Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679
Liu Y, Mochizuki K, Gorovsky MA (2004) Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena. Proc Natl Acad Sci U S A 101:1679–1684
Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297:1833–1837
Lim AK, Tao L, Kai T (2009) piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline. J Cell Biol 186:333–342
Klenov MS, Lavrov SA, Stolyarenko AD, Ryazansky SS, Aravin AA, Tuschl T, Gvozdev VA (2007) Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline. Nucleic Acids Res 35:5430–5438
Shpiz S, Olovnikov I, Sergeeva A, Lavrov S, Abramov Y, Savitsky M, Kalmykova A (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39:8703–8711
Jackson DA, Iborra FJ, Manders EM, Cook PR (1998) Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol Biol Cell 9:1523–1536
Sigova A, Vagin V, Zamore PD (2006) Measuring the rates of transcriptional elongation in the female Drosophila melanogaster germ line by nuclear run-on. Cold Spring Harb Symp Quant Biol 71:335–341
Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322:1845–1848
Rougvie AE, Lis JT (1988) The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell 54:795–804
Acknowledgments
This work was supported by grants to A.K. from the Russian Academy of Sciences program for Molecular and Cell Biology and the Russian Foundation for Basic Researches (12-04-00797).
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Shpiz, S., Kalmykova, A. (2014). Analyses of piRNA-Mediated Transcriptional Transposon Silencing in Drosophila: Nuclear Run-On Assay on Ovaries. In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_12
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DOI: https://doi.org/10.1007/978-1-62703-694-8_12
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Publisher Name: Humana Press, Totowa, NJ
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