NMD: a multifaceted response to premature translational termination

Abstract

Although most mRNA molecules derived from protein-coding genes are destined to be translated into functional polypeptides, some are eliminated by cellular quality control pathways that collectively perform the task of mRNA surveillance. In the nonsense-mediated decay (NMD) pathway premature translation termination promotes the recruitment of a set of factors that destabilize a targeted mRNA. The same factors also seem to have key roles in repressing the translation of the mRNA, dissociating its terminating ribosome and messenger ribonucleoproteins (mRNPs), promoting the degradation of its truncated polypeptide product and possibly even feeding back to the site of transcription to interfere with splicing of the primary transcript.

Figure 1. Structures and consequences of interactions between factors involved in NMD

a Interactions between the Upf proteins. Yeast amino acid numbering has been used to define the interaction domains and the size of the respective proteins. RNP= putative ribonucleoprotein domain, MIF4G= middle portion of eIF4G, CH= cysteine- and histidine-rich zinc-finger domain, 1B, 1C= additional regulatory domains. b Upf2 interaction switches Upf1 from closed (left) to open (right) conformation. The respective structures are of yeast Upf1 and human Upf1:Upf2, with coloring as indicated in the linear Upf1 model depicted in a. Yeast Upf1 (left) is shown bound to RNA-ADP:AIF4- (black). The Upf1:Upf2 heterodimer did not associate with RNA under the experimental conditions used for structure determination. Modified with permission from^,.

Figure 2. Activation of metazoan NMD by EJC-dependent interactions

The exon-junction complex (EJC) is a group of proteins deposited on an mRNA during splicing, 20-24 nt 5’ of an exon-exon boundary^,,,. The composition of the EJC is dynamic, and includes at least the core proteins Y14, Magoh, Barentz, and eIF4AIII, and one effector of NMD, Upf3^,,,-. In mammalian cells, Upf3 is loaded onto mRNAs during splicing and binds to a composite site comprised of parts of Y14, Magoh, and eIF4AIII. Upf2 is thought to join the complex in the cytoplasm via Upf3 binding, after mRNA export from the nucleus (top image). In parallel, Upf1 associates with eRF1-bound eRF3, and Smg-1, Smg-8, and Smg-9 associate with Upf1, collectively forming the SURF complex^,. If premature translational termination leads to retention of the downstream EJC then Upf1:Upf2 interaction is facilitated, leading to the formation of the DECID complex and to Upf1 phosphorylation and ATPase activation (middle image). Activation of NMD independently of Upf2, Upf3, or some EJC components has been described, suggesting that alternative pathways may also exist^,. Upf1 phosphorylation inhibits translation in cis and promotes its interaction with Smg-6, an endonuclease that can cleave the mRNA, and with the Smg-5:Smg-7 complex, which appears to promote deadenylation and decapping (bottom image)^,,,,,,. Mago=Magoh; BTZ=Barentz; R1=eRF1; R3=eRF3. Modified with permission from

Figure 3. Alternative models for NMD activation by premature termination

Not all mRNAs require an EJC for NMD activation, particularly in lower eukaryotes. The Upf proteins may associate with a prematurely terminating ribosome because essential interactions between Pab1 (or other 3’-UTR-associated proteins) and eRF3 have been disrupted (a), the mRNP context is non-accommodating for termination, i.e., critical proteins have not been added to or removed from the mRNP (a variation of the EJC model) (b), or the inefficiency of premature termination has left the ribosome in an atypical conformation (c). In all three situations the altered state is thought to allow Upf1 association with the eRFs and the ribosome, followed shortly by binding of the Upf2:Upf3 complex to Upf1 and the activation of mRNA decay.

Figure 4. Ancillary processes that accompany NMD

The association of the Upf proteins with a prematurely terminating ribosome has multiple consequences for the expression of the respective mRNA, including: A, translational repression of the mRNA, most likely at the level of initiation; B, disassembly of the mRNP and dissociation of the inefficiently terminating ribosome; C₁, accelerated decapping and/or poly(A) shortening of the targeted mRNA or C₂, endonucleolytic cleavage of the targeted mRNA; D, degradation of the nascent polypeptide; and E, feedback to the site of transcription, leading to the inhibition of splicing of the nascent pre-mRNA. The order of the different events is arbitrary, but is intended to imply that translational repression and mRNP disassembly may be logical prerequisites to the onset of mRNA decay. 4E=eIF4E; P=proteasome; AAA=poly(A) tail; R1=eRF1; R3=eRF3; 4G=eIF4G; 3=eIF3; 4A=eIF4A; structure marked “C”=Ccr4/Not deadenylase complex; Dcp1/Dcp2=decapping enzyme.

Figure 5. After interaction with the NMD and Smg factors nonsense-containing mRNAs are degraded by conventional cellular mRNA decay pathways

The nuclear biogenesis (upper left) and cytoplasmic translation and decay (center) of normal cellular mRNAs are depicted. Degradation of these mRNAs is initiated by exonucleolytic shortening of the poly(A) tail, catalyzed by the Ccr4:Caf1 and Pan2:Pan3 complexes^,,- (complexes not shown), or by endonucleolytic cleavage. Subsequent to poly(A) shortening, mRNAs are usually degraded by the decapping-dependent 5’ to 3’ pathway or by the 3′ to 5′ exosome-dependent pathway. The latter pathways are also utilized to eliminate the 5’ and 3’ mRNA fragments resulting from endonucleolytic cleavage. Nonsense-containing mRNAs recruit the Upf factors (or the Upf and Smg factors) to the prematurely terminating ribosome, resulting in interactions that promote accelerated entry of the mRNA into the poly(A)-shortening or decapping pathways, or lead to endonucleolytic cleavage. Products of endonucleolytic are then degraded by the standard 5’ to 3’ and 3’ to 5’ pathways. Some steps of the 5’ to 3’ pathway, including translational repression, may take place in cytoplasmic P-bodies (GW-bodies in mammalian and Drosophila cells; not shown), sites where the Dcp1/Dcp2 decapping enzyme, the Xrn1 5’-3’ exonuclease, and the Pat1, Dhh1, and Lsm1–7 decapping activators can accumulate^,. Evidence showing that decapped decay intermediates can be polyribosome-associated suggests that assembly of these structures is not essential for mRNA decay, at least in yeast. Moreover, in both yeast and metazoan cells, disruption of P bodies by depletion of core P body components failed to alter decay phenotypes for several different mRNAs^,,.