A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA Decay

Abstract

Eukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.

Keywords: mRNA degradation; mRNA surveillance; natural NMD targets; nonsense-mediated mRNA decay (NMD); nonstop decay (NSD); quality control.

Figure A1

Real-time PCR (RT-qPCR) analysis of human β-globin mRNAs variants in HeLa cells transiently co-transfected with Luciferase (LUC) siRNA (Control). The y-axis represents mRNA fold change. The control (Luciferase KD) was normalized to a baseline of 1, and all other conditions are shown relative to this baseline. Values above 1 indicate upregulation, while values below 1 indicate downregulation. Asterisks (*) indicate statistical significance relatively to the mRNA levels of the corresponding NMD target at control conditions. ** p < 0.01.

Figure 1

Human ribonucleases modulate the mRNA levels of different natural NMD targets. (A) Representative Western blot analysis of the UPF1, XRN1, DIS3, and DIS3L1 proteins and semiquantitative PCR analysis of the SMG6 and PM/Scl100 mRNA levels extracted from HeLa cells with (+) or without (−) knockdown (KD), to monitor KD efficiency. (B–G) Real-time PCR (RT-qPCR) analysis of natural NMD targets in HeLa cells transiently transfected with Luciferase (LUC) siRNA (Control), UPF1, SMG6, PM/Scl100, DIS3, DIS3L1, or XRN1 siRNAs. The y-axis represents fold change, with a value of 1 indicating no change relative to the control. Values above 1 indicate upregulation, while values below 1 indicate downregulation. The control (Luciferase KD) was normalized to a baseline of 1, and all other conditions are shown relative to this baseline. Asterisks (*) indicate statistical significance relative to the mRNA levels of the corresponding NMD target at control conditions: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 2

Human ribonucleases are involved in normal mRNA turnover as well as in the mRNA surveillance mechanisms of NMD and NSD. Representative semiquantitative PCR analysis of the SMG6 (A) or PM/Scl100 (B) mRNAs levels extracted from HeLa cells with (+) or without (−) knockdown (KD) and transiently transfected with plasmid-expressing wild type (βWT), NMD-resistant (β15), NMD-sensitive (β26 and β39), and NSD-sensitive (βNS) human β-globin mRNAs to monitor KD efficiency. (C–E) Representative Western blot analysis of protein extracted from HeLa cells with (+) or without (-) DIS3 and DIS3L1 KD and XRN1, respectively, and transiently co-transfected with plasmid-expressing βWT, β15, β26, β39, and βNS human β-globin mRNAs to monitor KD efficiency. (A–E) Real-time PCR (RT-qPCR) analysis of human β-globin mRNAs variants in HeLa cells transiently co-transfected with Luciferase (LUC) siRNA (Control), SMG6, PM/Scl100, DIS3, DIS3L1, or XRN1 siRNAs. The y-axis represents the fold change in mRNA levels. Values above 0 indicate upregulation, while values below 0 indicate downregulation. To compare the impact of each ribonuclease knockdown (KD) relative to the control, the fold change observed under Luciferase KD was subtracted from the fold change observed under each ribonuclease KD. This adjustment highlights the additional effect of the ribonuclease KD on mRNA variations compared to the control condition. Asterisks (*) indicate statistical significance relative to the mRNA levels of the corresponding NMD target under control conditions: * p < 0.05 and **** p < 0.0001.