mRNA surveillance of expressed pseudogenes in C. elegans

Messenger RNAs (mRNAs) that contain premature translation termination codons (PTCs) are targeted for rapid degradation in all eukaryotes tested. The mechanisms of nonsense-mediated mRNA decay (NMD) have been described in considerable detail, but the biological roles of NMD in wild-type organisms are poorly understood. mRNAs of wild-type organisms known to be degraded by NMD ("natural targets" of NMD) include by-products of regulated alternative splicing, out-of-frame mRNAs derived from unproductive gene rearrangements, cytoplasmic pre-mRNAs, endogenous retroviral and transposon RNAs, and mRNAs having upstream open reading frames or other unusual sequence features. NMD may function to eliminate aberrant PTC-containing mRNAs in order to protect cells from expression of potentially deleterious truncated proteins. Pseudogenes are nonfunctional genes or gene fragments that accumulate mutations through genetic drift. Such mutations will often introduce shifts of reading frame and/or PTCs, and mRNAs of expressed pseudogenes may thus be substrates of NMD. We demonstrate that mRNAs expressed from C. elegans pseudogenes are degraded by NMD and discuss possible implications for both mRNA surveillance and protein evolution. We describe an expressed pseudogene that encodes a small nucleolar RNA (snoRNA) within an intron and suggest this represents an evolutionary intermediate between snoRNA-encoding host genes that do or do not encode proteins.     

Regulation of nonsense-mediated mRNA decay: Implications for physiology and disease

Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that destabilizes aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes. This raises the possibility that NMD itself is under regulatory control. Indeed, several recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including microRNA circuits and NMD itself. Cellular stress also modulates the magnitude of NMD by mechanisms that are beginning to be understood. Here, we review the evidence that NMD is regulated and discuss the physiological role for this regulation. We propose that the efficiency of NMD is altered in some cellular contexts to regulate normal biological events. In disease states—such as in cancer—NMD is disturbed by intrinsic and extrinsic factors, resulting in altered levels of crucial NMD-targeted mRNAs that lead to downstream pathological consequences. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

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.     

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.     

Targeting of aberrant mRNAs to cytoplasmic processing bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.     

The multiple lives of NMD factors: balancing roles in gene and genome regulation

Nonsense-mediated mRNA decay (NMD) largely functions to ensure the quality of gene expression. However, NMD is also crucial to regulating appropriate expression levels for certain genes and for maintaining genome stability. Furthermore, just as NMD serves cells in multiple ways, so do its constituent proteins. Recent studies have clarified that UPF and SMG proteins, which were originally discovered to function in NMD, also have roles in other pathways, including specialized pathways of mRNA decay, DNA synthesis and cell-cycle progression, and the maintenance of telomeres. These findings suggest a delicate balance of metabolic events - some not obviously related to NMD - that can be influenced by the cellular abundance, location and activity of NMD factors and their binding partners.     

无义介导的mRNA降解(NMD)

无义介导的mRNA降解(nonsense-mediated mRNA decay,NMD）作为真核细胞中重要RNA监控机制,识别并降解开放阅读框中含有提前终止密码子（premature termination codon,PTC）的mRNA,以避免因截短的蛋白产物积累对细胞造成毒害. NMD还调控正常生理基因的表达,暗示其在真核细胞中扮演重要角色. NMD途径的关键是PTC的识别.本文通过3种模型来分别阐述发现于哺乳动物、酵母等不同有机体的识别机制.通常由NMD因子UPF1（up-frameshift）等被招募至含PTC的mRNA上,借助这些因子组装形成“功能复合体”并激活降解.但目前对于PTC识别后的过程仍认识有限,本文通过综述NMD途径的分子机制以更好地理解其生物学意义.     

Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

Background

Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2.

Results

We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation.

Conclusions

Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.