Lynne E. Maquat

Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics

Studies of nonsense-mediated mRNA decay in mammalian cells have proffered unforeseen insights into changes in mRNA–protein interactions throughout the lifetime of an mRNA. Remarkably, mRNA acquires a complex of proteins at each exon–exon junction during pre-mRNA splicing that influences the subsequent steps of mRNA translation and nonsense-mediated mRNA decay. Complex-loaded mRNA is thought to undergo a pioneer round of translation when still bound by cap-binding proteins CBP80 and CBP20 and poly(A)-binding protein 2. The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.

The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions

Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half‐life. There is accumulating evidence that pre‐mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of ∼335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20–24 nucleotides upstream of exon–exon junctions. Splicing‐dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing‐associated factors SRm160, DEK and RNPS1, the mRNA‐associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense‐mediated mRNA decay pathway, are discussed.

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3′-untranslated regions (3′ UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3′ UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3′ UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3′ UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).

Regulation of cytoplasmic mRNA decay

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means of modulating gene expression and thereby protein production. Up until recently, studies largely focused on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay and the ribonucleases that catalyse decay. Now, current studies have begun to elucidate how the decay process is regulated. This Review examines our current understanding of how mammalian cell mRNA decay is controlled by different signalling pathways and lays out a framework for future research.

Quality Control of mRNA Function

While individual nuclear and cytoplasmic reactions required for the formation of functional mRNA can be carried out in isolation in vitro, it has become increasingly clear that many of the steps along the path from gene to protein are, in vivo, interdependent in a way that provides important mechanisms for the QC of mRNA function. In fact, nothing less would be expected of an efficiently operating assembly line, which should discard defective products rather than proceed to process them. Owing to space and reference constraints, this minireview describes neither the bulk of data demonstrating the interdependence of reactions required for mRNA biosynthesis, nor the finer details of these reactions. Future work will no doubt reveal the complete network of integrated events that ensures mRNA QC and yet-to-be-defined molecular constituents of this network.‡E-mail: lynne_maquat@urmc.rochester.edu (L. E. M.); gcarmich@neuron.uchc.edu (G. G. C.).

Nonsense-mediated mRNA decay in mammals

Nonsense-mediated mRNA decay (NMD) in mammalian cells generally degrades mRNAs that terminate translation more than 50-55 nucleotides upstream of a splicing-generated exon-exon junction (reviewed in [Maquat, 2004a][1]; [Nagy and Maquat, 1998][2]). Notably, dependence on exon-exon junctions

The exon junction complex is detected on CBP80‐bound but not eIF4E‐bound mRNA in mammalian cells: dynamics of mRNP remodeling

Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon–exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense‐mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap‐binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap‐binding protein eIF4E. These and other data indicate that NMD targets CBP80‐bound mRNA during a ‘pioneer’ round of translation, but whether nuclear eIF4E also binds nascent but dead‐end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron‐containing RNA and the C‐terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80‐bound but not eIF4E‐bound mRNA. Each of these proteins is also detected on CBP80‐bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.

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.

At least one intron is required for the nonsense-mediated decay of triosephosphate isomerase mRNA: a possible link between nuclear splicing and cytoplasmic translation.

ABSTRACT Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3′-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3′-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3′-most intron from pre-mRNA “marks” the 3′-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5′ untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

Upf1 Phosphorylation Triggers Translational Repression during Nonsense-Mediated mRNA Decay

In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.