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

Szczegóły
Abstrakt

Tytuł:: sRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay.
Autorzy:: Tristan-Ramos P; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.; Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain.
Morell S; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.
Sanchez L; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.
Toledo B; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.; Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain.
Garcia-Perez JL; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.; MRC-Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.
Heras SR; Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, GENYO, Granada, Spain.; Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain.
Źródło:: Philosophical transactions of the Royal Society of London. Series B, Biological sciences [Philos Trans R Soc Lond B Biol Sci] 2020 Mar 30; Vol. 375 (1795), pp. 20190346. Date of Electronic Publication: 2020 Feb 10.
Typ publikacji:: Journal Article; Research Support, Non-U.S. Gov't
Język:: English
Imprint Name(s):: Original Publication: London : Royal Society, 1934-
MeSH Terms:: Genetic Techniques*
Long Interspersed Nucleotide Elements*
Retroelements*
MicroRNAs/*genetics
RNA, Small Interfering/*genetics
Cell Culture Techniques
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Grant Information:: United Kingdom WT_ Wellcome Trust; IECS-55007420 United States HHMI Howard Hughes Medical Institute
Contributed Indexing:: Keywords: Fanconi anaemia; LINE-1; cell culture-based retrotransposition reporter assay; miR-20; miRNAs; siRNAs
Molecular Sequence:: figshare 10.6084/m9.figshare.c.4796259
Substance Nomenclature:: 0 (MicroRNAs)
0 (RNA, Small Interfering)
0 (Retroelements)
Entry Date(s):: Date Created: 20200221 Date Completed: 20210121 Latest Revision: 20210401
Update Code:: 20240105
PubMed Central ID:: PMC7061984
DOI:: 10.1098/rstb.2019.0346
PMID:: 32075559
: Czasopismo naukowe

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'.

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