sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay

Author:

Tristan-Ramos Pablo12,Morell Santiago1,Sanchez Laura1,Toledo Belen12,Garcia-Perez Jose L.13ORCID,Heras Sara R.12ORCID

Affiliation:

1. Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain

2. Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain

3. MRC-Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK

Abstract

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’.

Funder

Secretaría de Estado de Investigación, Desarrollo e Innovación

Wellcome Trust University of Edinburgh

FP7 Ideas: European Research Council

Howard Hughes Medical Institute

Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía

Publisher

The Royal Society

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology

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