Journal of Molecular Biology
Volume 426, Issue 22, 11 November 2014, Pages 3689-3702
Journal home page for Journal of Molecular Biology

Communication
A Highly Conserved Region Essential for NMD in the Upf2 N-Terminal Domain

https://doi.org/10.1016/j.jmb.2014.09.015Get rights and content

Highlights

  • What is the role of Upf2 N-terminal domain in NMD pathway?

  • Determination of the structure of mIF4G-1 domain from yeast Upf2.

  • Upf2 mIF4G-1 domain harbors a highly conserved region mandatory for NMD.

  • This region seems to be involved in protein recruitment.

Abstract

Upf1, Upf2, and Upf3 are the principal regulators of nonsense-mediated mRNA decay (NMD), a cytoplasmic surveillance pathway that accelerates the degradation of mRNAs undergoing premature translation termination. These three proteins interact with each other, the ribosome, the translation termination machinery, and multiple mRNA decay factors, but the precise mechanism allowing the selective detection and degradation of nonsense-containing transcripts remains elusive. Here, we have determined the crystal structure of the N-terminal mIF4G domain from Saccharomyces cerevisiae Upf2 and identified a highly conserved region in this domain that is essential for NMD and independent of Upf2's binding sites for Upf1 and Upf3. Mutations within this conserved region not only inactivate NMD but also disrupt Upf2 binding to specific proteins, including Dbp6, a DEAD-box helicase. Although current models indicate that Upf2 functions principally as an activator of Upf1 and a bridge between Upf1 and Upf3, our data suggest that it may also serve as a platform for the association of additional factors that play roles in premature translation termination and NMD.

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.

References (69)

  • C. Mazza et al.

    Crystal structure of the human nuclear cap binding complex

    Mol Cell

    (2001)
  • J. Basquin et al.

    Architecture of the nuclease module of the yeast Ccr4-not complex: the Not1-Caf1-Ccr4 interaction

    Mol Cell

    (2012)
  • N. Fukuhara et al.

    SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway

    Mol Cell

    (2005)
  • B.S. Strunk et al.

    A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

    Cell

    (2012)
  • T.D. Goddard et al.

    Visualizing density maps with UCSF Chimera

    J Struct Biol

    (2007)
  • W. Wriggers et al.

    Situs: a package for docking crystal structures into low-resolution maps from electron microscopy

    J Struct Biol

    (1999)
  • M. Graille et al.

    Surveillance pathways rescuing eukaryotic ribosomes lost in translation

    Nat Rev Mol Cell Biol

    (2012)
  • S. Kervestin et al.

    NMD: a multifaceted response to premature translational termination

    Nat Rev Mol Cell Biol

    (2012)
  • C.J. Shoemaker et al.

    Translation drives mRNA quality control

    Nat Struct Mol Biol

    (2012)
  • Y. Cui et al.

    Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon

    Genes Dev

    (1995)
  • P. Leeds et al.

    The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon

    Genes Dev

    (1991)
  • K. Czaplinski et al.

    The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs

    Genes Dev

    (1998)
  • F. He et al.

    Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen

    Genes Dev

    (1995)
  • P.V. Ivanov et al.

    Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways

    EMBO J

    (2008)
  • E.E. Min et al.

    Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

    RNA

    (2013)
  • K.D. Swisher et al.

    Interactions between Upf1 and the decapping factors Edc3 and Pat1 in Saccharomyces cerevisiae

    PLoS One

    (2011)
  • H. Chamieh et al.

    NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity

    Nat Struct Mol Biol

    (2008)
  • K. Czaplinski et al.

    Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation

    RNA

    (1995)
  • F. He et al.

    Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway

    Mol Cell Biol

    (1997)
  • L. Aravind et al.

    Eukaryote-specific domains in translation initiation factors: implications for translation regulation and evolution of the translation system

    Genome Res

    (2000)
  • M. Clerici et al.

    Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2

    Nucleic Acids Res

    (2014)
  • J. Kadlec et al.

    The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3

    Nat Struct Mol Biol

    (2004)
  • G. Serin et al.

    Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4)

    Mol Cell Biol

    (2001)
  • A. Grimson et al.

    SMG-1 is a phosphatidylinositol kinase-related protein kinase required for nonsense-mediated mRNA decay in Caenorhabditis elegans

    Mol Cell Biol

    (2004)
  • Cited by (0)

    Z.F. and B.R. contributed equally to this work.

    View full text