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Article

AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites

Structure et Instabilité des Génomes, Muséum National d’Histoire Naturelle, CNRS UMR 7196, INSERM U1154, 43 rue Cuvier, F-75005 Paris, France
*
Author to whom correspondence should be addressed.
Present address: Genetic and Epigenetic Alterations of Genomes, de Duve Institute, Faculty of Pharmacy and Biomedical Sciences, Université Catholique de Louvain, 1200 Brussels, Belgium.
Biomolecules 2020, 10(6), 827; https://doi.org/10.3390/biom10060827
Received: 7 April 2020 / Revised: 12 May 2020 / Accepted: 21 May 2020 / Published: 28 May 2020
(This article belongs to the Special Issue Epigenetics in Cancer)
Alternative promoter usage involved in the regulation of transcription, splicing, and translation contributes to proteome diversity and is involved in a large number of diseases, in particular, cancer. Epigenetic mechanisms and cis regulatory elements are involved in alternative promoter activity. Multiple transcript isoforms can be produced from a gene, due to the initiation of transcription at different transcription start sites (TSS). These transcripts may not have regions that allow discrimination during RT-qPCR, making quantification technically challenging. This study presents a general method for the relative quantification of a transcript synthesized from a particular TSS that we called AP-TSS (analysis of particular TSS). AP-TSS is based on the specific elongation of the cDNA of interest, followed by its quantification by qPCR. As proof of principle, AP-TSS was applied to two non-coding RNA: telomeric repeat-containing RNAs (TERRA) from a particular subtelomeric TSS, and Alu transcripts. The treatment of cells with a DNA methylation inhibitor was associated with a global increase of the total TERRA level, but the TERRA expression from the TSS of interest did not change in HT1080 cells, and only modestly increased in HeLa cells. This result suggests that TERRA upregulation induced by global demethylation of the genome is mainly due to activation from sites other than this particular TSS. For Alu RNA, the signal obtained by AP-TSS is specific for the RNA Polymerase III-dependent Alu transcript. In summary, our method provides a tool to study regulation of gene expression from a given transcription start site, in different conditions that could be applied to many genes. In particular, AP-TSS can be used to investigate the epigenetic regulation of alternative TSS usage that is of importance for the development of epigenetic-targeted therapies. View Full-Text
Keywords: alternative TSS; alternative promoter; TERRA; transcript isoform; transcript quantification; Alu alternative TSS; alternative promoter; TERRA; transcript isoform; transcript quantification; Alu
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MDPI and ACS Style

Le Berre, G.; Hossard, V.; Riou, J.-F.; Guieysse-Peugeot, A.-L. AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites. Biomolecules 2020, 10, 827. https://doi.org/10.3390/biom10060827

AMA Style

Le Berre G, Hossard V, Riou J-F, Guieysse-Peugeot A-L. AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites. Biomolecules. 2020; 10(6):827. https://doi.org/10.3390/biom10060827

Chicago/Turabian Style

Le Berre, Gabriel, Virginie Hossard, Jean-Francois Riou, and Anne-Laure Guieysse-Peugeot. 2020. "AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites" Biomolecules 10, no. 6: 827. https://doi.org/10.3390/biom10060827

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