Post-transcriptional Inhibition of Luciferase Reporter Assays by the Nod-like Receptor Proteins NLRX1 and NLRC3
Abstract
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.
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BibTeX
@article{ling2026posttranscriptional,
title = {{Post-transcriptional Inhibition of Luciferase Reporter Assays by the Nod-like Receptor Proteins NLRX1 and NLRC3}},
author = {Arthur Ling and Fraser Soares and David Croitoru and Ivan Tattoli and Leticia A.M. Carneiro and Michele Boniotto and Szilvia Benko and Dana Philpott and Stephen Girardin},
journal = {Journal of Biological Chemistry},
year = {2026},
month = oct,
doi = {10.1074/jbc.m111.333146},
url = {https://hal.science/hal-05797399v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Post-transcriptional Inhibition of Luciferase Reporter Assays by the Nod-like Receptor Proteins NLRX1 and NLRC3
AU - Arthur Ling
AU - Fraser Soares
AU - David Croitoru
AU - Ivan Tattoli
AU - Leticia A.M. Carneiro
AU - Michele Boniotto
AU - Szilvia Benko
AU - Dana Philpott
AU - Stephen Girardin
PY - 2026
DA - 2026/10/13
JO - Journal of Biological Chemistry
DO - 10.1074/jbc.m111.333146
UR - https://hal.science/hal-05797399v1
AB - 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.
SN - 0021-9258
SN - 1083-351X
ER -