sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay
| Autor: | Jose L. Garcia-Perez, Sara R. Heras, Belen Toledo, Laura Sánchez, Pablo Tristan-Ramos, Santiago Morell |
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| Rok vydání: | 2020 |
| Předmět: |
Transposable element
Small RNA Retroelements Cell Culture Techniques Retrotransposon Computational biology Biology General Biochemistry Genetics and Molecular Biology LINE-1 03 medical and health sciences 0302 clinical medicine microRNA RNA Small Interfering 030304 developmental biology Ribonucleoprotein Regulation of gene expression 0303 health sciences Reporter gene Articles siRNAs Long interspersed nuclear element MicroRNAs Long Interspersed Nucleotide Elements Genetic Techniques miRNAs Fanconi anaemia miR-20 General Agricultural and Biological Sciences cell culture-based retrotransposition reporter assay 030217 neurology & neurosurgery |
| Zdroj: | Philos Trans R Soc Lond B Biol Sci |
| ISSN: | 1471-2970 0962-8436 |
| Popis: | The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’. |
| Databáze: | OpenAIRE |
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