Chapter 4 NMD: Multitasking Between mRNA Surveillance and Modulation of Gene Expression
Introduction
After 15 years or so of a somewhat shadowy existence, nonsense‐mediated mRNA decay (NMD) research has only recently left this sheltered realm and has developed into one of the hottest fields both in molecular and cellular biology and in molecular medicine. Both—the previously arcane life of NMD and the present surge of interest also from unexpected directions—are fuelled by the amazing features of this complex and still enigmatic mechanism and its molecular actors. Early on, nonsense mutations were recognized to be linked to or to underlie many human disease phenotypes, yet the most frequent impact of nonsense mutations often escaped (and still escapes) many researchers: contrary to intuition and to the most prevalent interpretation, a premature termination codon (PTC), that is a stop codon within an open reading frame (ORF), does not usually induce the synthesis of a truncated protein to which the observed phenotype could be, and often is, ascribed; instead, it triggers the decay of the mutated mRNA. This phenomenon—that cells already monitor the quality of gene expression products at intermediate steps—is astounding in itself but the way it is enacted is even more prodigous.
To discriminate between termination codons that specify the end of an ORF and those that interrupt it, mammals interlock two pivotal yet spatially separate processes in gene expression: nuclear splicing and cytoplasmic translation. Interestingly, the details differ in various organisms yet the phenomenon of NMD itself is common to all metazoans, thus underscoring its vital significance. Yet, the differences in the details are significant and stress the importance of screening the DNA and protein databases not only for similarities as the most important functional features because evolution has treasured them but also because it is as much difference as kinship that matters in evolution.
NMD is complex. This basic property is evident in a multitude of aspects. First, on the biochemical level, NMD is complex because it combines both nuclear and cytoplasmic events. In addition, NMD seems to dispose of several entrance gates that guide potential substrates to degradation and for these options it uses common yet also divergent sets of molecules. In that respect, NMD may turn out to be paradigmatic for many other cellular processes; cellular systems increasingly present themselves as densely meshed, interactive, dynamic, and flexible networks rather than as linear pathways in which one step has to be completed before the next can occur. It looks as if gene expression is better described as a fluid rather than a stepwise process. Therefore, NMD could serve as a model for the elicudation of principles that also underly many other poorly understood mechanisms; its study could help to purge the too prevalent mechanical perceptions of biochemical processes that have been borrowed from nineteenth‐century industry.
Second, NMD is complex in that it not only serves the recognition and elimination of faulty transcripts but also appears to modulate the expression of a plethora of physiological transcripts. These physiological substrates have one feature in common with their pathological counterparts: they possess a termination codon that is, by NMD standards, conceived as premature. This applies, for example, to the termination codons of upstream ORFs, to termination codons that are followed by splice events in the 3′ untranslated region (UTR), or to termination codons that are introduced into an ORF as the result of somatic DNA rearrangements, alternative splicing, ribosomal frameshifting, or mRNA editing. In some cases, these features are exploited for self‐regulatory mechanisms. For example, when a gene product induces the alternative splicing of its own transcript, a PTC may be introduced into its ORF or a splice junction may be generated 3′ to the termination codon, thus directing the resulting alternative transcript to NMD. Moreover, it is suspected that potentially NMD‐sensitive physiological transcripts can stand at crossing points of pathways or networks and thus modulate such pathways as a whole. This, of course, implies that such a potential “regulator transcript” at the center of a pathway can be either exposed to or concealed from NMD or that NMD itself is regulated so that the putative regulator at times escapes NMD and at others is downmodulated by it. There are indications that both of these options occur.
Third, NMD is complex because with respect to its role in human disease, it is janus‐faced. It can be beneficial for heterozygous carriers of nonsense mutations because it destroys the product of the faulty allele and prevents dominant negative impacts of truncated proteins. Yet, it can also result in the clinical picture of protein deficiency in those cases where it prevents the production of a protein that, albeit truncated, would convey at least a residual function.
Fourth, the intricacy of NMD is enhanced in that it emerges as a biological multiplayer. Single or cohorts of its effectors or maybe even itself as a whole seem to moonlight both in other branches of mRNA metabolism and in apparently unrelated functions.
This chapter presents the current state of knowledge about the mechanism of nonsense‐mediated mRNA decay and its molecular actors. Moreover, it tries to illuminate the intricate network of contacts with other cellular pathways and to pinpoint the switch of function of several of its effectors that moonlight in these other pathways. Finally, it highlights the importance of NMD for the understanding of many opaque genotype–phenotype relationships of human hereditary and acquired disease conditions.
Section snippets
General rules
NMD specifically recognizes and degrades mRNAs with PTCs and thus protects the cell from potentially harmful C‐terminally truncated polypeptides. To accomplish the task of discrimination between premature and physiological termination codons, NMD uses a complicated and stunning network composed of early and late elements of gene expression (reviewed in Conti and Izaurralde, 2005, Hentze and Kulozik, 1999, Lejeune and Maquat, 2005, Maquat, 2004). Premature termination or nonsense codons can be
Targets of NMD
The class of NMD substrates that first comes to mind is mRNAs with an erroneous in‐frame stop codon that interrupts their ORF. PTCs can be acquired by nonsense and frameshift mutations. Furthermore, unproductive somatic DNA rearrangements [in the case of T‐cell receptor (TCR) and immunoglobulin genes], transcriptional errors, and splice errors can result in frameshifts and subsequently in reading frames that terminate prematurely. Other types of faulty transcripts are exemplified by pre‐mRNAs
Disobedient NMD Targets
According to current mechanistic understanding in mammalia, PTCs that reside >50 nt 5′ to the last exon–exon junction should elicit NMD. Consistent with and obedient to this rule, mRNAs transcribed from intronless genes and transcripts with PTCs that reside beyond this border are NMD resistant (Brocke et al., 2002, Maquat and Li, 2001, Neu‐Yilik et al., 2001). However, the picture is more complicated than these results imply. The sensitivity of transcripts to NMD varies widely. As previously
Good Cop, Bad Cop: Medical Importance of NMD
Genetic disorders are commonly caused by nonsense or frameshift mutations that introduce PTCs, and NMD helps to avoid the production of large amounts of C‐terminally truncated peptides. The medical importance of NMD is well documented in β‐thalassemia, which exemplifies the phenotypic impact of the polar effect of PTC mutations at different positions within the same gene (Holbrook et al., 2004 and references therein). If PTC mutations are located at positions that activate NMD, the common
Moonlighting NMD Proteins: Moonlighting Pathway?
NMD factors either alone or in teams entertain a multitude of contacts to other cellular pathways and functions. These range from an as yet poorly understood role of Upf1, Smg‐5, and Smg‐6 in RNAi of C. elegans (Domeier et al., 2000) and of Upf1 in RNAi in plants (Arciga‐Reyes et al., 2006), over roles of various NMD factors in translation, UPF1 functions in nonsense‐associated altered splicing (NAS) (Mendell et al., 2002, Zhang and Krainer, 2006) and in other mRNA turnover pathways, and tasks
Conclusions
Substantial progress has been made in the last decade to elucidate the mechanism of NMD, the factors involved, and their mode of action, as well as to identify pathological and physiological NMD targets. Moreover, the understanding of the multiple functions of NMD has been considerably deepened. However, as is customary in science, each step forward confronts us with new and surprising enigmas. There are several burning issues to tackle. (1) It is an unsolved and important question whether, as
Acknowledgments
The authors thank Jill Holbrook for critical reading of the manuscript and the members of the Kulozik laboratory for insightful and inspiring discussions.
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