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PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response.
Max Myakishev, Oksana Polesskaya,
Valentina Kulichkova, Ancha Baranova, Larissa Gause, Irina Konstantinova

This study has been published: Cell Stress Chaperones. 2008 Spring;13(1):111-6.

Purpose: It has been previously shown that short interspersed repetitive elements (SINEs) behave as stress-inducible genes transcribed by Pol III. SINE transcription could be induced in cultured cells and in living animals
by various stress treatments including heat shock, viral infection and translational inhibition. DNA-dependent RNA polymerases II (Pol II) and III (Pol III) are different in their cellular localizations and functions. All classical protein-coding genes are transcribed by Pol II, whereas ibosomal 5S RNA, tRNA, other small RNAs, and most of the repetitive elements, including SINEs, are transcribed by Pol III. The majority of nearly 30,000 Pol II-transcribed genes covering 5% of the genome are represented by unique or low-copy number genes. Transposable elements transcribed
by Pol III, on the contrary, cover much larger fraction of the genome and are numbered in millions. Differentiated cells of the adult mammal transcribe only a small fraction of their Pol II- and Pol III-dependent genes.The rest of the genome is silenced by DNA methylation and modification of histones. This silencing of Pol III transcription of SINEs is relieved in response to stress viral infectionsm, tumorogenesis, and organogenesis.

Experimental Design and Results: Until now, Pol III transcripts of SINEs have been detected and quantified by the primer extension method, which utilize a reverse-transcription reaction primed by a radioactively labeled oligonucleotide located in the body of a SINE with subsequent separation of resulting cDNAs in acrylamide gels. In gels, a Pol III-generated cDNA band is separated from the Pol II-generated smear as the former is homogeneous and shorter than Pol II transcripts. Here we propose a novel method that is based on adaptor ligation followed by PCR, more sensitive and allows high- throughput real-time PCR format. Therefore, this approach should facilitate the research of transposable elements.

Fig. 1. Principle of specific amplification of Pol III-generated transcripts of transposon B1 by adaptor-PCR method.

Experimental applications of the adaptor-PCR method: To test this method, we chose a model of the rodent cell response to UV stress and studied Pol III-dependent transcription of transposon B1, a rodent homolog of human Alu repeat. In the first experiment, we demonstrated that Pol III- dependent transcription of transposon B1 is affected by UVB irradiation, a strong genotoxic cellular stress.

In another experiment, we applied this method to the study of subcelullar localization of Pol III transcripts of transposon B1 in rat livers.we demonstrated an enrichment of the Pol III-dependent transcripts in the nucleus.

Conclusion: we developed an adaptor-PCR-based method for detection of Pol III transcripts that should facilitate the research of transposable elements, particularly in the context of cellular stress.