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

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.

Dynamic silencing of somatic L1 retrotransposon insertions reflects the developmental and cellular contexts of their genomic integration

“…Moreover, upon integration they could trigger antisense transcripts [ 19 ] or otherwise artificially trigger or disrupt silencing by mimicking actively transcribed, protein-coding gene. A recent study noted that differences in promoters, reporter sequences or integration sites could influence reporter expression and thereby affect the study conclusions [ 78 ].…”

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

“…We used a protocol adapted from [60], where we first transfected siRNAs and then co-transfected siRNAs with L1 reporter plasmids. Besides functional validation of siRNAs, it is important to note that some 'non-targeting control' siRNAs (NTCs) are known to interfere with L1 retrotransposition, and their use should be avoided [61].…”

“…Thus, we next optimized the L1 retrotransposition reporter protocol using siRNAs. Notably, a previous study demonstrated that widely used non-targeting control siRNAs (NTCs) could interfere with L1 retrotransposition [61]. The mechanism of the interference is unknown, and the use of these NTC siRNA controls should be avoided, at least in combination with the L1-retrotransposition reporter assay (see Methods for further details).…”