Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

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 homeostasis governed by cell type-specific and branched feedback loops acting on NMD

Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.     

RNA splicing promotes translation and RNA surveillance

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.     

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.     

Context analysis of termination codons in mRNA that are recognized by plant NMD

The nonsense-mediated mRNA decay (NMD) system is an RNA surveillance system that degrades mRNAs possessing premature translation termination codons (PTCs). Although NMD factors are well conserved in eukaryotes, it is speculated that the contexts of those termination codons that are subject to NMD are different depending on the organism. Context analysis of termination codons that are recognized by the plant NMD system would clarify NMD target mRNAs in plants, and contribute to our understanding of its biological relevance in plants. In the present study we analyzed the positions of termination codons that were recognized as PTCs using an Agrobacterium transient expression assay, i.e. the accumulation of a series of plant mRNAs with nonsense mutations in different contexts was tested in plants. The results indicated that termination codons that are located distant from the mRNA 3' termini or >50 nucleotides upstream of the 3'-most exon-exon junction are recognized as substrates for NMD.