Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition.

G. Travis Tabor,Pamela R. Cook

doi:10.1186/s13100-016-0079-3

Abstract

BackgroundThe Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.ResultsWe found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.ConclusionsEngineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

Highlights

The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome
We report here that: 1) different populations of HeLa cells can result in different experimental outcomes; 2) two presumably complementary L1 retrotransposition reporter assays produced conflicting results when coupled with exogenously expressed p38δ; and 3) two different non-targeting control (NTC) small interfering RNA sequences differentially affected measured L1 activity
mitogen-activated protein kinases (MAPKs) p38δ phosphorylates ORF1p on S/T-P motifs We first determined whether activated wild type p38δ (WT, Invitrogen) could phosphorylate ORF1p on its S/ T-P motifs, which are required for robust L1 activity [31]

Summary

Introduction

The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. We recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). The only active, autonomous mobile DNA element in humans is the Long INterspersed Element-1 (LINE-1, L1) retrotransposon, which is responsible for generating almost half of the human genome via insertion of its own DNA and that of non-autonomous Short-INterspersed repeat Elements (SINES) [1]. These insertions, combined with 3′ transductions, nonallelic homologous recombination and mobilization of cellular mRNAs, have had a. The expression of one isoform, p38δ, can be induced in primary cell cultures via exogenous expression of ORF1p [34]

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