In a series of three recent publications [9
] Steve Brenner and colleagues suggested, by data-mining, that one-third of reliably inferred human splice products form a major class of natural targets for NMD. Alternative splicing is thought to occur in 30-60% of human genes, expanding the coding repertoire of the limited number of genes in the genome and modulating tissue-specific and developmental gene functions. Brenner and colleagues [9
] now suggest that alternative splicing provides a mechanism to generate PTC-containing splice products that are subsequently degraded by NMD and, as a consequence, that cooperation between alternative splicing and NMD pathways offers a major and currently underappreciated way to regulate gene expression.
For their in silico
analysis, they mapped well-characterized human RefSeq [16
] and LocusLink [17
] mRNA sequences to genomic sequences, and then performed high-stringency alignments between these 'RefSeq-coding genes' and expressed sequence tags (ESTs). The reliably inferred splice variants were only accepted as likely NMD targets when they conformed to the '50-nucleotide rule': this hallmark of mammalian NMD predicates that stop codons located at least 50 nucleotides upstream of the last exon junction will be interpreted as 'premature' and trigger NMD. This approach leads to an underestimate of potential NMD targets, because some PTCs (for example, in T-cell receptor gene transcripts) will trigger NMD even when they are not followed by a sufficiently distant intron [18
]. Moreover, Brenner and colleagues [9
] also excluded mRNA variants that are indistinguishable from products of faulty splicing. These studies have unearthed several groups of functionally related proteins whose expression appears to be regulated by NMD, including translation factors and ribosomal proteins. This is in remarkable contrast to the yeast data of He et al
], where proteins with a function in translation were, if anything, underrepresented in the pool of NMD-regulated genes.
NMD is not (yet) on everyone's mind. As a consequence, Brenner and colleagues [11
] found several entries for truncated proteins in the Swiss-Prot database. In some of these cases the available experimental evidence confirms that the mRNAs that encode these truncated proteins are bona fide
NMD substrates. We are left with a consolation and a surprise. The consolation is that traces of NMD can be uncovered even though they had been overlooked before. Having recently had to accept that the number of genes in the human genome is too limited to explain the far higher number of proteins (not to mention other gene products), we then had to learn that one plausible and elegant explanation lies in alternative splicing, which enables a gene to code for a whole family of related, or sometimes antagonistic, proteins. And now the surprise is that a large portion of this effort is supposedly expended only to direct many of the primary products to decay. Several conclusions can be drawn from these observations. First, databases need to be read and annotated with a full realization of the implications of NMD; and second, NMD seems to serve as a tool for rapidly switching off gene expression. This view extends the idea of NMD as a mechanism for ridding the cell of the potentially harmful products of faulty splicing. But there may be more to it. NMD rarely downregulates the expression of a transcript completely; more commonly, 10-30% of the PTC-containing transcripts survive and may allow the production of physiologically relevant levels of truncated protein products.
For NMD researchers it has always been hard to reconcile these observations with the presumed protective role of NMD, especially as very low levels of biological products can sometimes have enormous effects. Given the problems of detecting the low levels of proteins or peptides produced from downregulated transcripts, in addition to some lingering lack of awareness of NMD, examples to prove otherwise are hard to come by. A recent publication [19
] describes a PTC-containing transcript of the high-affinity immunoglobulin E (IgE) receptor, FcεRIβ, arising from retention of an intron. This alternative transcript not only conforms to the 50-nucleotide rule but its expression levels are very low compared to those of the full-length transcript, as would be expected for an NMD target. Nonetheless, the truncated protein is not only detectable, it even competes effectively with the full-length protein to control FcεRIβ expression on the cell surface. Thus, even low endogenous expression levels of NMD targets can suffice to generate a product with a biological function. A similar example of the utility of a bona fide
NMD substrate is illustrated by the unc-49
locus in C. elegans
. This locus uses alternative splicing to produce three GABA-receptor subunits, two of which (A and B) undergo several splice events in their 3' UTRs, rendering them predicted NMD substrates [20
]. While A-form transcripts are hardly detectable, B-form transcripts represent the most abundant form and code for a protein essential for the worm's locomotion. Either the B-form transcript escapes NMD, or the residual mRNA left after NMD suffices for the necessary protein production.
Among the examples presented by Brenner and colleagues [11
] or in other studies [21
] are genes with complex alternative splice patterns resulting in multiple transcript isoforms with or without PTCs. The lower abundance of these isoforms when compared to the full-length form supports the notion that they are targeted to the NMD pathway. But is the possibility that cells engage in complex alternative-splicing procedures generating multiple products, just to finally dispose of them, the only conceivable option? Four out of five PTC-containing isoforms of the mRNA for the LARD death receptor are readily detectable in non-activated lymphocytes, whereas only the full-length form is expressed in activated lymphocytes [11
]. While it is entirely possible that the PTC-containing forms are generated to switch off receptor expression in resting lymphocytes, there are attractive alternatives: as in the case of the FcεRIβ receptor [19
], the PTC-containing mRNAs may produce proteins or peptides with relevant, although currently unknown, functions. Thus, quantitative control of the expression of low amounts of protein isoforms could represent yet another facet of the function of the NMD pathway.
A role for NMD in controlling the levels of noncoding RNAs (including noncoding alternative splice products) must also be considered. RNA accounts for more than 95% of the human genome's output and there is increasing evidence that noncoding RNAs (including introns, and spliced and polyadenylated transcripts) can have a function, for example as a modulating network or an additional layer of information [22
]. Importantly, noncoding RNAs have been discovered among natural NMD targets in yeast [8
], where only a relatively small portion of the genome is transcribed into noncoding RNAs. What might be the role of NMD in mammals, which transcribe a far higher percentage of their genome into noncoding RNAs [22
]? Clearly, the recent insights into the RNA-substrate spectrum of the NMD system should enhance the appreciation of NMD as a versatile, multipurpose mechanism that controls the transcriptome qualitatively and quantitatively.