Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife

Nonsense-mediated mRNA decay (NMD) downmodulates mRNAs that have in-frame premature termination codons and prevents translation of potentially harmful truncated proteins from aberrant mRNAs. Two new approaches have identified physiological NMD substrates, and suggest that NMD functions as a multipurpose tool in the modulation of gene expression.     

RNA surveillance. Unforeseen consequences for gene expression, inherited genetic disorders and cancer

Messenger RNAs are monitored for errors that arise during gene expression by a mechanism called RNA surveillance, with the result that most mRNAs that cannot be translated along their full length are rapidly degraded. This ensures that truncated proteins are seldom made, reducing the accumulation of rogue proteins that might be deleterious. The pathway leading to accelerated mRNA decay is referred to as nonsense-mediated mRNA decay (NMD). The proteins that catalyze steps in NMD in yeast serve two roles, one to monitor errors in gene expression and the other to control the abundance of endogenous wild-type mRNAs as part of the normal repertoire of gene expression. The NMD pathway has a direct impact on hundreds of genetic disorders in the human population, where about a quarter of all known mutations are predicted to trigger NMD.     

The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression

Proper neuronal function and several forms of synaptic plasticity are highly dependent on precise control of mRNA translation, particularly in dendrites. We find that eIF4AIII, a core exon junction complex (EJC) component loaded onto mRNAs by pre-mRNA splicing, is associated with neuronal mRNA granules and dendritic mRNAs. eIF4AIII knockdown markedly increases both synaptic strength and GLUR1 AMPA receptor abundance at synapses. eIF4AIII depletion also increases ARC, a protein required for maintenance of long-term potentiation; arc mRNA, one of the most abundant in dendrites, is a natural target for nonsense-mediated decay (NMD). Numerous new NMD candidates, some with potential to affect synaptic activity, were also identified computationally. Two models are presented for how translation-dependent decay pathways such as NMD might advantageously function as critical brakes for protein synthesis in cells such as neurons that are highly dependent on spatially and temporally restricted protein expression.     

Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly.     

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

Involvement of SR proteins in mRNA surveillance

Nonsense mutations influence several aspects of gene expression, including mRNA stability and splicing fidelity, but the mechanism by which premature termination codons (PTCs) can apparently affect splice-site selection remains elusive. We used a model human beta-globin gene with duplicated 5' splice sites (5'ss) and found that PTCs inserted between the two 5'ss do not directly influence splicing in this system. Instead, their apparent effect on 5'ss selection in vivo is an indirect result of nonsense-mediated mRNA decay (NMD), as conditions that eliminated NMD also abrogated the effect on splicing. Remarkably, we found an unexpected function of SR proteins in targeting several mRNAs with PTCs to the NMD pathway. Overexpression of various SR proteins strongly enhanced NMD, and this effect required an RS domain. Our data argue against a universal role of PTCs in regulating pre-mRNA splicing and reveal an additional function of SR proteins in eukaryotic gene expression.