Compound C inhibits nonsense-mediated RNA decay independently of AMPK

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development

Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe

Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct—we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages

Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process

Inhibition of Nonsense-Mediated mRNA Decay by Antisense Morpholino Oligonucleotides Restores Functional Expression of hERG Nonsense and Frameshift Mutations in Long-QT Syndrome

Mutations in the human ether-a-go-go-related gene (hERG) cause long-QT syndrome type 2 (LQT2). We previously described a homozygous LQT2 nonsense mutation Q1070X in which the mutant mRNA is degraded by nonsense-mediated mRNA decay (NMD) leading to a severe clinical phenotype. The degradation of the Q1070X transcript precludes the expression of truncated but functional mutant channels. In the present study, we tested the hypothesis that inhibition of NMD can restore functional expression of LQT2 mutations that are targeted by NMD. We showed that inhibition of NMD by RNA interference-mediated knockdown of UPF1 increased Q1070X mutant channel protein expression and hERG current amplitude. More importantly, we found that specific inhibition of downstream intron splicing by antisense morpholino oligonucleotides prevented NMD of the Q1070X mutant mRNA and restored the expression of functional Q1070X mutant channels. The restoration of functional expression by antisense morpholino oligonucleotides was also observed in LQT2 frameshift mutations. Our findings suggest that inhibition of NMD by antisense morpholino oligonucleotides may be a potential therapeutic approach for some LQT2 patients carrying nonsense and frameshift mutations

Position of Premature Termination Codons Determines Susceptibility of hERG Mutations to Nonsense-Mediated mRNA Decay in Long QT Syndrome

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC)mutations by nonsense-mediatedmRNA decay (NMD) is an importantmechanismof long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts asNMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to thewild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs*25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned N54–60 nt upstream of the 3′-most exon–exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078*. The present study defines the positional requirements for the susceptibility of LQT2mutations toNMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD

Developments in non-mandatory disclosures in annual reports of companies: A case study

The paper investigates the extent of non-mandatory disclosure of information (NMD) in the annual reports of the 17 companies listed on the South Pacific Stock Exchange (SPSE) in Fiji, a developing country, and whether NMD by these companies has changed over time providing additional and useful information to stakeholders. The empirical data was gathered from the years 2008 to 2010 to provide a clear picture of the change in the level and extent of NMD, and its influences over the periods 2008 to 2010. It can be seen from the Fiji perspective that the mandatory requirements tend to have a financial focus. However, it would be expected that the level of company disclosures would have changed over time, with not only global market forces but through differing societal values which have increased the frequency and demand of non-mandatory reporting by companies. All companies showed some degree of NMD, and on average this demonstrates an increasing trend. The stakeholders are receiving more information about a company’s activities. The companies were analysed in light of recent developments in corporate governance by the Capital Markets Development Authority (CMDA) implementing their 10 corporate governance principles. This became a major driver of the increase in NMD levels of the disclosures in the annual reports of the listed companies. However, a large variation still exists between the level and extent of the NMD and the different listed companies. The minimum disclosure level found over the three years was 9.09 percent, which has increased to a minimum of 13.66 percent in 2010, and the maximum disclosure level over the three years was 81.82 percent. The findings for the extent of NMD was also similar where the minimum words used in NMDs was 114, increasing to 854 in 2010, and the maximum disclosure extent over the three years was 21,414 words. However, it was found that the measurement of counting words tended to fluctuate over different periods where significant events took place that affected the company. Therefore, it was established that disclosure is impacted by what happens in the reporting period, and can explain why one period may have greater disclosure than another. The paper aims to extend earlier work of Sharma & Davey (2013) on the extent of NMD in Fijian context. While Sharma & Davey (2013) considered voluntary disclosure from 1999-2005, our study reviews NMD over 2008-2010. The study has shown that corporate governance code issued in 2009 by Capital Market Development Authority has influenced the level of NMD