A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

Graphical Abstract

Figure 1.

The EJC-independent and EJC-enhanced reporter system design. (A) Top: The NMD+: EJC-independent, NMD+: EJC-enhanced, and NMD– reporter designs, as well as the theoretical mRNAs and proteins that the plasmid reporters can produce. Bottom: The theoretical RNA and protein levels expected from the reporters for NMD and its associated phenomena. (B) Experimental workflow for these reporters as well as possible assays to measure RNA and protein levels. R1 and R2 are either BFP and RFP (respectively), or Renilla and Firefly luciferase (respectively) depending on fluorescent or luminescent readouts. R3 is always GFP.

Figure 2.

The EJC-independent and EJC-enhanced reporter system enables quantitative investigation of variable NMD efficiency. RNA levels for fluorescent reporters (A) and luminescent reporters (B) as measured by RT-qPCR. Protein levels for fluorescent reporters (C) measured by flow cytometry and plotted as a bar graph using the median RFP intensities. Protein levels for luminescent reporters (D) as measured by luciferase assays. FF refers to Firefly luciferase. Transfections were performed 3 times and the mean values of RNA and protein levels were plotted in the bar graphs. Dots overlaid on each bar indicate the individual data points for that sample. Error bars reflect the standard error of the mean. Student's t-tests were conducted to determine statistical significance (*p< 0.05, **p< 0.005, ***p< 0.0005).

Figure 3.

Genomically integrated NMD reporters show less cell-to-cell variability in NMD activity than transiently expressed reporters. (A) Schematic of stable integration using the HILO-RMCE method described by Khandelia et al., figure adapted from Khandelia et al. (56). (B) RNA levels for stably integrated fluorescent reporters as measured by RT-qPCR. (C) Protein levels for stably integrated fluorescent reporters measured by flow cytometry and plotted as a bar graph using the median RFP intensities. Data obtained from three technical replicates of stably integrated cell lines and the mean values of RNA and protein levels were plotted in bar graphs. Dots overlaid on each bar indicate the individual data points for that sample. Error bars reflect the standard error of the mean. Student's t-tests were conducted to determine statistical significance (*p< 0.05, **p< 0.005, ***p< 0.0005). (D) Flow cytometry histograms showing the RFP distribution of BFP+ cells for NMD+: EJC-independent, NMD+: EJC-enhanced, and NMD– reporters, when transiently transfected or stably integrated. The three shades of gray (DMSO treatment) or red (SMG1i treatment) represent different replicates.

Figure 4.

EJC-independent and EJC-enhanced reporters respond differentially when treated with readthrough inducing drugs. (A) Top: schematic of NMD+ and readthrough control. R1 is BFP, R2 is RFP, and R3 is GFP. Bottom: Representative flow diagrams for each reporter. The black arrow is pointing to the quadrant of interest which shows the percentage of cells that are GFP positive. (B) Percentage of GFP positive cells for NMD positive reporters when treated with DMSO, G418, SMG1i, and SMG1i + G418. (C) GFP protein levels normalized to BFP for each of the reporters when treated with DMSO G418, SMG1i, and SMG1i + G418. (D) RFP protein levels normalized to BFP for each of the reporters when treated with DMSO, G418, SMG1i, and SMG1i + G418. Transfections were performed 3 times and the mean values plotted in the bar graphs. Dots overlaid on each bar indicate the individual data points for that sample. Error bars reflect the standard error of the mean. Student's t-tests were conducted to determine statistical significance (*p< 0.05, **p< 0.005, ***p< 0.0005, ****p< 0.00005).

Figure 5.

The NMD reporter landscape. A summary of existing NMD reporter systems, their ability to test various aspects of NMD and their experimental compatibilities. Each reporter system includes the NMD+ reporter and its NMD– control. Fluorescent or luminescent reporter genes are represented by colored boxes (CBG: Click Beetle Green luciferase, CBR: Click Beetle Red luciferase), gray boxes represent exons of known NMD targets, and black carets indicate their introns. Various promoter elements driving reporter gene expression are shown as gray boxes with arrows, and orange lines denote PTCs. Testable aspects of NMD includes to the two branches of NMD and the NMD associated mechanisms PTC readthrough and truncated protein degradation. Experimental compatibilities cover stable recombinase-mediated genomic integration, massively parallel reporter assays, and inducibility if the reporter has an inducible promoter. Filled gray circles indicate fulfillment of a criterion, while empty gray circles indicate conditional fulfillment. The Sato and Singer reporters are conditional for readthrough because GFP protein levels cannot distinguish between readthrough and truncated protein expression. The Baker and Hogg reporters are only suitable for massively parallel screening if they can be stably integrated. The reporters introduced in this study are highlighted with a red box.