Post-transcriptional inhibition of luciferase reporter assays by the Nod-like receptor proteins NLRX1 and NLRC3.
Luciferase reporter assays (LRAs) are widely used to assess the activity of specific signal transduction pathways. Although powerful, rapid and convenient, this technique can also generate artifactual results, as revealed for instance in the case of high throughput screens of inhibitory molecules. Here we demonstrate that the previously reported inhibitory effect of the Nod-like receptor (NLR) protein NLRX1 on NF-κB- and type I interferon-dependent pathways in LRAs was a nonspecific consequence of the overexpression of the NLRX1 leucine-rich repeat (LRR) domain. By comparing luciferase activity and luciferase gene expression using quantitative PCR from the same samples, we showed that NLRX1 inhibited LRAs in a post-transcriptional manner. In agreement, NLRX1 also repressed LRAs if luciferase was expressed under the control of a constitutive promoter, although the degree of inhibition by NLRX1 seemed to correlate with the dynamic inducibility of luciferase reporter constructs. Similarly, we observed that overexpression of another NLR protein, NLRC3, also resulted in artifactual inhibition of LRAs; thus suggesting that the capacity to inhibit LRAs at a post-transcriptional level is not unique to NLRX1. Finally, we demonstrate that host type I interferon response to Sendai virus infection was normal in NLRX1-silenced human HEK293T cells. Our results thus highlight the fact that LRAs are not a reliable technique to assess the inhibitory function of NLRs, and possibly other overexpressed proteins, on signal transduction pathways.
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M6G 2T6, Canada.
Arthur
A
Ling
Fraser
F
Soares
David O
DO
Croitoru
Ivan
I
Tattoli
Leticia A M
LA
Carneiro
Michele
M
Boniotto
Szilvia
S
Benko
Dana J
DJ
Philpott
Stephen E
SE
Girardin
D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
D036341:Intercellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D016328:NF-kappa B; C503285:NLRC3 protein, human; C527006:NLRX1 protein, human; D011994:Recombinant Proteins; D008156:Luciferases