Abstract
Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5′ ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.
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Abbreviations
- Pol:
-
polymerase
- SINE:
-
short interspersed nuclear element
- UV:
-
ultraviolet
- UTR:
-
untranslated region
- PA:
-
polyacrylamide
References
Allen TA, Von Kaenel S, Goodrich JA, Kugel JF (2004) The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nat Struct Mol Biol 11:816–821
Bohlander SK, Espinosa R 3rd, Le Beau MM, Rowley JD, Diaz MO (1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics 13:1322–1324
Borodulina OR, Kramerov DA (1999) Wide distribution of short interspersed elements among eukaryotic genomes. FEBS Lett 457:409–413
Borodulina OR, Kramerov DA (2005) PCR-based approach to SINE isolation: simple and complex SINEs. Gene 349:197–205
Carey MF, Singh K, Botchan M, Cozzarelli NR (1986) Induction of specific transcription by RNA polymerase III in transformed cells. Mol Cell Biol 6:3068–3076
de Gruijl FR (2002) Photocarcinogenesis: UVA vs. UVB radiation. Skin Pharmacol Appl Skin Physiol 15:316–320
Dlugosz AA, Glick AB, Tennenbaum T, Weinberg WC, Yuspa SH (1995) Isolation and utilization of epidermal keratinocytes for oncogene research. Methods Enzymol 254:3–20
Grigoryan MS, Kramerov DA, Tulchinsky EM, Revasova ES, Lukanidin EM (1985) Activation of putative transposition intermediate formation in tumor cells. Embo J 4:2209–2215
Hasler J, Samuelsson T, Strub K (2007) Useful ‘junk’: Alu RNAs in the human transcriptome. Cell Mol Life Sci 64:1793–1800
Kimura RH, Choudary PV, Stone KK, Schmid CW (2001) Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes. Cell Stress Chaperones 6:263–272
Konstantinova IM, Kozlov Yu V, Kulitchkova VA, Petukhova OA (1988) Small cytoplasmic RNA associated with polyadenylated RNA is involved in the hormonal regulation of gene expression. FEBS Lett 238:320–324
Kramerov DA, Lekakh IV, Samarina OP, Ryskov AP (1982) The sequences homologous to major interspersed repeats B1 and B2 of mouse genome are present in mRNA and small cytoplasmic poly(A) + RNA. Nucleic Acids Res 10:7477–7491
Lander ES, Linton LM, Birren B et al (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921
Li T, Spearow J, Rubin CM, Schmid CW (1999) Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo. Gene 239:367–372
Liu WM, Chu WM, Choudary PV, Schmid CW (1995) Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts. Nucleic Acids Res 23:1758–1765
Maraia RJ, Driscoll CT, Bilyeu T, Hsu K, Darlington GJ (1993) Multiple dispersed loci produce small cytoplasmic Alu RNA. Mol Cell Biol 13:4233–4241
Meunier JR, Sarasin A, Marrot L (2002) Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing. Photochem Photobiol 75:437–447
Rubin CM, Kimura RH, Schmid CW (2002) Selective stimulation of translational expression by Alu RNA. Nucleic Acids Res 30:3253–3261
Rudin CM, Thompson CB (2001) Transcriptional activation of short interspersed elements by DNA-damaging agents. Genes Chromosomes Cancer 30:64–71
Russanova VR, Driscoll CT, Howard BH (1995) Adenovirus type 2 preferentially stimulates polymerase III transcription of Alu elements by relieving repression: a potential role for chromatin. Mol Cell Biol 15:4282–4290
Schmid CW (1996) Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog Nucleic Acid Res Mol Biol 53:283–319
Tao K, Li J, Warner J, MacLeod K, Miller FR, Sahagian GG (2001) Multiple lysosomal trafficking phenotypes in metastatic mouse mammary tumor cell lines. Int J Oncol 19:1333–1339
Taylor KD, Piko L (1987) Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos. Development 101:877–892
White RJ (2004) RNA polymerase III transcription and cancer. Oncogene 23:3208–3216
Wick N, Luedemann S, Vietor I et al (2003) Induction of short interspersed nuclear repeat-containing transcripts in epithelial cells upon infection with a chicken adenovirus. J Mol Biol 328:779–790
Acknowledgement
The authors thank Dr. Carl Schmid for contributing an idea of adaptor ligation as a means of detection of Pol III transcripts. This work was supported by the grant 050449606 from the Russian Fund for Basic Research (RFBR), the Program MCB RAS and performed in the Institute of Cytology, St. Petersburg, and the Koltsov Institute of Developmental Biology, Moscow, Russia.
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Myakishev, M., Polesskaya, O., Kulichkova, V. et al. PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response. Cell Stress and Chaperones 13, 111–116 (2008). https://doi.org/10.1007/s12192-008-0010-z
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DOI: https://doi.org/10.1007/s12192-008-0010-z