Impact Of Nonsense-mediated mRNA Decay Research Articles

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021

The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy.

Premature termination codons (PTCs) can result in the production of truncated proteins or the degradation of mRNAs by nonsense-mediated mRNA decay (NMD). Which of these outcomes occurs can alter the effect of a mutation, with the engagement of NMD depending upon a series of rules. Here, by applying these rules genome-wide to obtain a resource called NMDetective, we explore the impact of NMD on genetic disease and approaches to therapy. First, human genetic diseases differ in whether NMD typically aggravates or alleviates the effects of PTCs. Second, failure to trigger NMD is a cause of ineffective gene inactivation by CRISPR-Cas9 gene editing. Finally, NMD is a determinant of the efficacy of cancer immunotherapy, with only frameshifted transcripts that escape NMD predicting a response. These results demonstrate the importance of incorporating the rules of NMD into clinical decision-making. Moreover, they suggest that inhibiting NMD may be effective in enhancing cancer immunotherapy.

The rules and impact of nonsense-mediated mRNA decay in human cancers.

Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-quarters of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification of tumor suppressor genes.

Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome

Nonsense and frameshift mutations are common in congenital long QT syndrome type 2 (LQT2). We previously demonstrated that hERG nonsense mutations cause degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD) and are associated with mild clinical phenotypes. The impact of NMD on the expression of hERG frameshift mutations and their phenotypic severity is not clear. The purpose of this study was to examine the role of NMD in the pathogenesis of a hERG frameshift mutation, P926AfsX14, identified in a large LQT2 kindred and characterize genotype-phenotype correlations. Genetic screening was performed among family members. Phenotyping was performed by assessment of ECGs and LQTS-related cardiac events. The functional effect of P926AfsX14 was studied using hERG cDNA and minigene constructs expressed in HEK293 cells. Significant cardiac events occurred in carriers of the P926AfsX14 mutation. When expressed from cDNA, the P926AfsX14 mutant channel was only mildly defective. However, when expressed from a minigene, the P926AfsX14 mutation caused a significant reduction in mutant mRNA, protein, and hERG current. Inhibition of NMD by RNA interference knockdown of up-frameshift protein 1 partially restored expression of mutant mRNA and protein and led to a significant increase in hERG current in the mutant cells. These results suggest that NMD is involved in the pathogenic mechanism of the P926AfsX14 mutation. Our findings suggest that the hERG frameshift mutation P926AfsX14 primarily results in degradation of mutant mRNA by the NMD pathway rather than production of truncated proteins. When combined with environmental triggers and genetic modifiers, LQT2 frameshift mutations associated with NMD can manifest with a severe clinical phenotype.

Widespread Impact of Nonsense-Mediated mRNA Decay on the Yeast Intronome

Nonsense-mediated mRNA decay (NMD) eliminates transcripts carrying premature translation termination codons, but the role of NMD on yeast unspliced pre-mRNA degradation is controversial. Using tiling arrays, we show that many unspliced yeast pre-mRNAs accumulate in strains mutated for the NMD component Upf1p and the exonuclease Xrn1p. Intron identity and suboptimal splicing signals resulting in weak splicing were found to be important determinants in NMD targeting. In the absence of functional NMD, unspliced precursors accumulate in the cytoplasm, possibly in P-bodies. NMD can also complement RNase III-mediated nuclear degradation of unspliced RPS22B pre-mRNAs, degrades most unspliced precursors generated by a 5' splice site mutation in RPS10B, and limits RPS29B unspliced precursors accumulation during amino acid starvation. These results show that NMD has a wider impact than previously thought on the degradation of yeast-unspliced transcripts and plays an important role in discarding precursors of regulated or suboptimally spliced transcripts.

Impact of Nonsense-Mediated mRNA Decay on the Global Expression Profile of Budding Yeast

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd+) and mutant (Nmd−) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% ± 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd− strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Impact of Nonsense-mediated mRNA Decay on the Global Expression Profile of Budding Yeast

Impact of Nonsense-mediated mRNA Decay on the Global Expression Profile of Budding Yeast

Cytoplasmic nonsense-mediated mRNA decay for a nonsense (W262X) transcript of the gene responsible for hereditary tyrosinemia, fumarylacetoacetate hydrolase

Messenger RNAs containing premature stop codons are generally targeted for degradation through the nonsense-mediated mRNA decay (NMD) pathway. The subcellular localization of the NMD process in higher eukaryotes remains controversial. While many mRNAs are subjected to NMD prior to their release from the nucleus, a few display cytoplasmic NMD. To understand the possible impact of NMD on the pathogenesis of hereditary tyrosinemia type I, a severe metabolic disease caused by fumarylacetoacetate hydrolase (FAH) deficiency, we examined the metabolism of FAH mRNA harboring a nonsense mutation, W262X, in lymphoblastoid cell lines derived from patients and their parents. W262X-FAH transcripts show a ∼20-fold reduction in abundance in mutant cells, which is translation-dependent. Cellular fractionation shows that this down-regulation of the W262X transcript occurs in the cytoplasm. Thus, the W262X FAH is another example of nonsense mRNAs subjected to the NMD pathway in the cytoplasm.