Journal of Molecular Biology
CommunicationA Highly Conserved Region Essential for NMD in the Upf2 N-Terminal Domain
Graphical Abstract
Introduction
Nonsense-mediated mRNA decay (NMD) is one of three conserved eukaryotic surveillance pathways ensuring mRNA quality control in the cytoplasm [1], [2], [3], [4]. NMD is activated by mechanistic differences between normal and premature translation termination and utilizes three conserved factors (Upf1, Upf2, and Upf3) [5], [6], [7] to couple nonsense codon recognition to the release factors (eRF1 and eRF3), the ribosome, and the mRNA decapping complex [8], [9], [10], [11], [12], [13], [14]. Upf1, the central regulator of NMD [2], encompasses an N-terminal zinc knuckle CH domain and a functional helicase core domain exhibiting RNA binding and ATPase activities [15], [16]. The Upf1 CH domain binds to the C-terminal region of Upf2, the largest component of the Upf1–Upf2–Upf3 surveillance complex [17]. The N-terminal two-thirds of Upf2 include three domains that adopt the same fold as the middle domain of eukaryotic initiation factor eIF4G (mIF4G; Fig. 1a) [18], [19], [20], [21]. The third of these mIF4G domains (mIF4G-3) interacts with the central RNA recognition motif (RRM) from Upf3 [20]. Accordingly, Upf2 is thought to act as a scaffolding protein that bridges Upf1 and Upf3 [15], [17], [22]. Other factors are necessary for NMD in higher eukaryotes [5] and appear to regulate the activity or availability of the Upf proteins. Thus, in human, the SMG-1 and SMG-5 to SMG-9 proteins, as well as the PP2A phosphatase, act as NMD enhancers by regulating UPF1's phosphorylation status [23], [24], [25] and the exon junction complex (EJC) interacts with the human UPF3b isoform [26], [27].
Although multiple mechanistic models have been proposed for NMD in lower and higher eukaryotes [2], [28], [29], [30], [31], [32], [33], little is known about the precise role of the three Upf proteins in the individual steps of NMD: recognition of prematurely terminating mRNAs, targeting of an mRNA and its nascent polypeptide for accelerated degradation, and disassembly of the prematurely terminating mRNP complex. The most elaborate details uncovered to date pertain to Upf1–Upf2 interaction and the possible role of this interaction in activating Upf1's ATPase and helicase activities that are essential for NMD. Prior to interacting with Upf2, Upf1 adopts a closed conformation that appears to optimize its RNA binding capabilities while simultaneously minimizing its ATPase and helicase activities [15], [34]. Upf2 interaction with the Upf1 CH domain [35] triggers a major conformational change of the CH domain, opening the structure of Upf1 while simultaneously reducing its RNA-binding activity and stimulating its ATPase and helicase activities [34]. These effects are comparable to the consequences of deleting the Upf1 CH domain [15], [34]. While these results provide some insights into the function of Upf2's Upf1-interaction domain, they leave the role of a large fragment of Upf2 unresolved, namely, the N-terminal half encompassing the first and second mIF4G domains. Previous study has highlighted the critical role of the whole N-terminal domain and particularly the region spanning residues 30–50 of yeast Upf2 mIF4G1 domain for NMD [36]. Very recently, it was also shown that the deletion of the human UPF2 mIF4G-1 or mIF4G-2 domain, or both, completely inhibits NMD without affecting UPF2 protein stability or cellular localization or UPF1 and UPF3 recruitment [19]. However, tethering of these truncated UPF2 proteins to PTC-containing mRNA partially restores NMD, suggesting that the mIF4G-1 and mIF4G-2 UPF2 domains are important for recruitment of UPF2 to PTC-containing mRNAs [19]. Here, we have pursued structural and functional analyses of the role of the Upf2 mIF4G-1 domain in yeast NMD. Its crystal structure led to the characterization of a highly conserved region within this domain that is essential for NMD, as well as to the identification of proteins whose association with Upf2 is dependent on conserved residues within this domain. Our results suggest that Upf2 may have previously unanticipated mechanistic roles in NMD beyond serving as an activator of Upf1 activity or a bridge between Upf1 and Upf3.
Section snippets
Crystal structure of the S. cerevisiae Upf2 N-terminal domain
To investigate the biochemical and structural roles of the Upf2 N-terminal region in NMD, we cloned and expressed in Escherichia coli two fragments from Saccharomyces cerevisiae Upf2 (hereafter designated ScUpf2). One construct corresponds to the first N-terminal mIF4G domain (amino acids 1–360) while the other spans the three predicted Upf2 mIF4G domains (amino acids 1–820; Fig. 1a). Although both ScUpf2 fragments could be purified to homogeneity in large quantities, neither yielded
Discussion
Despite extensive studies within the last two decades, the mechanism by which NMD distinguishes nonsense-containing mRNAs and activates their accelerated decay remains elusive [2]. The Upf1, Upf2, and Upf3 proteins are central to NMD, and they interact with each other [17], the ribosome [13], [42], the eRF1 and eRF3 translation termination factors [9], [11], [51], [52], [53], and the mRNA decay machinery [10], [14]. Upf1 plays a pivotal role in NMD and its ATPase and RNA helicase activities are
Cloning, expression, and purification of Upf2 proteins
The sequence encoding C-terminally His-tagged Upf2 [1–310] was amplified from yeast S. cerevisiae S288C genomic DNA with oligonucleotides oMG27/oMG32 (see Table S1) and inserted into the pET21-a vector to yield plasmid pMG567. The DNA sequence encoding C-terminally His-tagged Upf2 [1–360] from S. cerevisiae was synthesized chemically by GenScript and cloned into pET21-a, yielding plasmid pMG464. The sequence encoding C-terminally His-tagged Upf2 [1–820] was amplified from yeast S. cerevisiae
Acknowledgments
We are indebted to Manuela Argentini and David Cornu (SICaps, IMAGIF platform, Gif/Yvette, France) and John Leszyk (UMass Medical School Proteomics and Mass Spectrometry Facility) for mass spectrometry and to Vonny Caroline for technical assistance. We thank SOLEIL for provision of synchrotron radiation facilities and Andrew Thompson for assistance with beamline Proxima-1. We acknowledge computing time at the Fundación Centro de Supercomputación de Castilla y León. We are indebted to Dr.
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Z.F. and B.R. contributed equally to this work.