Features and factors that dictate if terminating ribosomes cause or counteract nonsense-mediated mRNA decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a quality control pathway in eukaryotes that continuously monitors mRNA transcripts to ensure truncated polypeptides are not produced. The expression of many normal mRNAs that encode full-length polypeptides is also regulated by this pathway. Such transcript surveillance by NMD is intimately linked to translation termination. When a ribosome terminates translation at a normal termination codon, NMD is not activated, and mRNA can undergo repeated rounds of translation. On the other hand, when translation termination is deemed abnormal, such as that on a premature termination codon, it leads to a series of poorly understood events involving the NMD pathway, which destabilizes the transcript. In this review, we summarize our current understanding of how the NMD machinery interfaces with the translation termination factors to initiate NMD. We also discuss a variety of cis-acting sequence contexts and trans-acting factors that can cause readthrough, ribosome reinitiation, or ribosome frameshifting at stop codons predicted to induce NMD. These alternative outcomes can lead to the ribosome translating downstream of such stop codons and hence the transcript escaping NMD. NMD escape via these mechanisms can have wide-ranging implications on human health, from being exploited by viruses to hijack host cell systems to being harnessed as potential therapeutic possibilities to treat genetic diseases.

Keywords: nonsense mutations; nonsense-meditated mRNA decay; ribosome; translation; translation termination.

Figure 1

Key steps in normal translation termination and recycling. mRNA is shown in gray with the thicker line depicting the coding sequence and the thinner line depicting the “normal” 3′ UTR. The ribosome is shown in blue, and other termination factors are labeled. A, normal translation termination begins when eRF1 recognizes a termination codon (shown as UGA) and the following nucleotide (+4 position; preferred nucleotides are shown) in the A-site along with the GTP-bound form of eRF3 to form the termination complex. B, GTP hydrolysis by eRF3 causes a conformational change in eRF1 that positions it to mediate hydrolysis and release of the nascent polypeptide. C, eRF3-GDP dissociates from the termination complex, and ABCE1, bound to ATP, joins to begin ribosome recycling. D, ATP hydrolysis by ABCE1 leads to ribosome splitting followed by dissociation of eRF1, ABCE1, and 60S from the mRNA. E, the 40S recycling factor heterodimer MCT-1/DENR binds the terminated ribosome. F, the 40S subunit and P-site tRNA are then removed from the mRNA by MCT-1/DENR, freeing all ribosome components to engage in another round of translation.

Figure 2

NMD can occur in an EJC-dependent or EJC-independent manner. mRNA is shown in gray. The introduction of premature termination codon in coding sequence (thicker line) results in an “extended 3' UTR” that also includes the normal 3′ UTR (thinner line). The ribosome is shown in blue, and other important proteins are labeled. Recognition of NMD-inducing translation termination codon occurs through one of two general pathways: the EJC-independent (A) or EJC-dependent (B) pathway. A, the EJC-independent pathway begins when UPF1, the core NMD factor, is bound downstream of the terminating ribosome at abnormally high levels. After interaction between UPF1 and the termination complex, other NMD factors are recruited to assemble the “SURF” complex, which includes UPF1 phosphorylation factors SMG1 (the kinase) and its regulatory SMG8/9 heterodimer. This complex may include or subsequently recruit DHX34, an RNA helicase that may aid in interaction of UPF1 with its activators UPF2 and UPF3B (or its paralog UPF3A). Formation of the SURF complex leads to UPF1 phosphorylation (represented by yellow triangles labeled “P”) by SMG1. B, the EJC-dependent NMD requires the presence of an EJC downstream of the terminating ribosome that enhances NMD by recruiting UPF2 and UPF3B (or UPF3A) to mRNA. Following assembly of the SURF complex at the terminating ribosome and UPF1 interaction with its activators UPF2 and UPF3B (or UPF3A), which can be aided by DHX34, UPF1 gets phosphorylated by SMG1. C, after initial phosphorylation, UPF1 is phosphorylated more extensively. It is unknown how long the ribosome and release factors remain associated with the phosphorylated UPF1 (indicated by faded shapes). The extensive phosphorylation of UPF1 leads to the recruitment of SMG6 and SMG5/7 heterodimer, which initiate mRNA degradation via multiple mechanisms including SMG6-catalyzed mRNA endonucleolytic cleavage close to termination codons. EJC, exon junction complex; NMD, nonsense-mediated mRNA decay.

Figure 3

Interactions between UPF proteins and termination machinery.Left, a terminating ribosome. Right, a recycling ribosome. The UPF proteins interact with the ribosome and termination factors in both a direct (black lines) and indirect (purple lines) fashion. All three UPF proteins can interact with the 80S ribosome and the 40S subunit although the specific contacts within these assemblies are known only for UPF1. All three UPF proteins are also reported to interact with termination factors. The numbers on each arrow represent possible functions of each interaction listed in the legend below. References that provide evidence for the interactions between UPF proteins and termination machinery are included in parentheticals.

Figure 4

Stop codon readthrough can prevent NMD.Top, factors that promote stop codon readthrough: (1) the stop codon triplet used, where the UGA codon is the most permissive stop codon, (2) the identity of the nucleotide at +4 position where stop codon readthrough is promoted the most by a C, and (3) an AU-rich region downstream of the stop codon. Bottom, factors that inhibit stop codon readthrough: These are typically found adjacent to the normal termination codons and include a GC-rich region immediately downstream of the stop codon and the presence of poly-A binding protein 1 (PABP1) in spatial proximity of the termination codon. NMD, nonsense-mediated mRNA decay.

Figure 5

Ribosome reinitiation after a PTC can prevent NMD.A, when the ribosome terminates at a PTC, it is recognized by downstream UPF1, with the aid of the EJC, and the transcript undergoes NMD. See Figure 2 for more details. B, when ribosome recycling occurs poorly at a premature termination codon, for example, because of limiting recycling factors like ABCE1, this can lead to the ribosome reinitiation at a nearby downstream start codon. Reinitiation causes displacement of NMD activating UPF and EJC proteins, escape from NMD and production of an N-terminally truncated or a unique polypeptide. EJC, exon junction complex; NMD, nonsense-mediated mRNA decay; PTC, premature termination codon.