Nuclear Import of Upf3p Is Mediated by Importin- /- and Export to the Cytoplasm Is Required for a Functional Nonsense-Mediated mRNA Decay Pathway in Yeast

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin/ heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd phenotype. The addition of a functional NES element to an export-defective upf allele restores export and partially restores an Nmd phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone. EUKARYOTIC cells from a variety of organisms inet al. 1995; Zhang et al. 1995; Czaplinski et al. 1999). cluding yeast, nematodes, mice, and humans rapmRNAs containing a premature termination codon but idly eliminate mRNAs that contain a premature terminalacking a DSE fail to be degraded by the NMD pathway tion codon (Losson and Lacroute 1979; Leeds et al. (Peltz et al. 1993; Ruiz-Echevarria et al. 1996, 1998). 1991; Pulak and Anderson 1993; Perlick et al. 1996). In yeast, the three factors Upf1p, Upf2p, and Upf3p Nonsense mRNA degradation occurs through a pathway are required for NMD (Leeds et al. 1991, 1992; Cui et called nonsense-mediated mRNA decay (NMD), which al. 1995; He and Jacobson 1995; Lee and Culbertson serves two purposes. One is in RNA surveillance in which 1995). Mutations in the UPF genes stabilize nonsense nonsense mRNAs arising through errors in gene expresmRNAs, resulting in rates of decay similar to those of sion are rapidly eliminated to prevent the accumulation the corresponding wild-type mRNAs. In addition, the of deleterious truncated proteins (Pulak and Anderefficiency of translation termination at premature stop son 1993; Cali and Anderson 1998; Li and Wilkinson codons is decreased in strains carrying upf mutations 1998; Frischmeyer and Dietz 1999). A second purpose while the overall efficiency of nonsense mRNA translais to control the abundance of a subset of endogenous tion increases (Muhlrad and Parker 1999; Bidou et wild-type mRNAs containing built-in signals that can al. 2000). These effects, combined with the increase in trigger premature termination of translation, which mRNA stability, contribute to the ability of upf mutaleads to faster decay as part of the normal repertoire tions to suppress nonsense and frameshift mutations of gene expression (Lelivelt and Culbertson 1999). (Culbertson et al. 1980; Leeds et al. 1991, 1992; MadNMD requires a mechanism to distinguish a premature erazo et al. 2000). nonsense codon from the normal wild-type termination Homologs of the yeast Upf proteins have been identisignal. In yeast, this involves the presence of a degenerfied in Schizosaccharomyces pombe, Caenorhabditis elegans, ate downstream sequence element (DSE) located 3 of mice, and humans (Page et al. 1999; Lykke-Andersen a premature nonsense codon (Peltz et al. 1993; Hagan et al. 2000; Mendell et al. 2000; Aronoff et al. 2001; Serin et al. 2001). Human hUpf1p and hUpf2p were identified as homologs of yeast Upf1p and Upf2p (Per1These authors contributed equally to this work. lick et al. 1996; Applequist et al. 1997). A mutation in 2Corresponding author: Laboratory of Molecular Biology, R. M. Bock the conserved helicase domain of hUpf1p confers a Labs, University of Wisconsin, 1525 Linden Dr., Madison, WI 53706. E-mail: [email protected] dominant-negative phenotype in yeast (Leeds et al. Genetics 161: 1465–1482 (August 2002) 1466 R. L. Shirley et al. 1992) and partially inactivates the NMD pathway in Upf3p from the nucleus via a leucine-rich nuclear export sequence is required for a fully functional NMD monkey COS and human HeLa cells (Sun et al. 1998). Several homologs of yeast Upf3p were identified in hupathway. Our results support the hypothesis that Upf3p functions in one of the initial steps necessary to earmark mans (Lykke-Andersen et al. 2000; Serin et al. 2001). These proteins are derived from two genes, each of a nonsense mRNA for recruitment as a substrate in the NMD pathway. This occurs prior to or during export which expresses several isoforms due to alternative splicing. These studies suggest that the function of the Upf of a nonsense mRNA from the nucleus to the cytoplasm. proteins in identifying and targeting nonsense mRNAs for rapid decay is conserved among eukaryotes. MATERIALS AND METHODS Translation is required for the rapid decay of nonsense mRNAs. Nonsense mRNAs are stabilized by the Strains and plasmids: Strain RSy5 (MAT ade2-1 leu2-1 tyr7-1 can1-100 upf31 trp ura his3 GAL2 ) was used for immunopresence of nonsense tRNA suppressors (Losson and fluorescence microscopy and to assay suppression of the leu2-1, Lacroute 1979), and they are recruited into polyribotyr7-1, and can1-100 nonsense mutations. Strain LRSy323 somes (Leeds et al. 1991; He et al. 1993; Zhang et al. (MATa his4-38 SUF1-1 trp11 upf31 ura3-52 leu21) was 1997). In addition, a portion of the total cellular pool used for the experiment shown in Figure 1C. Strain PJ69-4A of Upf1p, Upf2p, and Upf3p cofractionates with polyri(MATa trp1-901 leu2-3,112 his3-200 gal4 gal80 LYS2::GAL1HIS3 GAL2-ADE2 met2::GAL7-lacZ; obtained from E. Craig) bosomes (Atkin et al. 1997). Physical interactions bewas used for the two-hybrid assay ( James et al. 1996). The tween the yeast Upf proteins suggest that they act in localization of Upf3p-HA was examined in strain PSY730 (MATa concert to promote NMD on polyribosomes (He and srp1-31 leu2-3,112 his3 ade2 trp1 ura3-52; Tabb et al. 2000; Jacobson 1995; He et al. 1997). The Upf proteins copurstrain obtained from P. Silver). The effects of CRM1 on Upf3p ify with release factor eRF3 and Upf1p also copurifies localization were examined in strains MNY12 (MATa CRM1:: Kan leu2 his3 trp1 ura3 [pDC-CRM1T539C-GFP]) and MNY8 with release factor eRF1 (Czaplinski et al. 1998; Wang (MATa CRM1::Kan leu2 his3 trp1 ura3 [pDC-CRM1T539Cet al. 2001). HA]; Neville and Rosbash 1999). The CRM1T529C allele Upf1p associates with polyribosomes in the absence confers sensitivity to leptomycin B. of Upf2p or Upf3p, and it appears to facilitate the Plasmids are listed in Table 1. All plasmids were created dissociation of Upf2p with polyribosomes. Upf3p is reusing techniques and reagents as described previously (Shirley et al. 1998). pAF8 was constructed by ligating the XhoIquired for the association of Upf2p with polyribosomes SacI fragment from pUZ178 (Atkin et al. 1997) containing (Atkin et al. 1997). On the basis of these results, it was UPF2 into the same sites in pRS423. Plasmids expressing green proposed that Upf3p recruits Upf2p to polyribosomes fluorescent protein (GFP) fusion constructs were made as (Atkin et al. 1997; Culbertson 1999, 2001). Upf2p follows. Oligomers (Operon, Alameda, CA) were designed to may then facilitate NMD by interacting with Upf1p (He amplify the UPF3 promoter and 5 -untranslated region (UTR), using pLS17 as a template. The product was subcloned into et al. 1996). Upf1p could be recruited to polyribosomes a shuttle vector using unique restriction sites engineered by through an association with release factors (He et al. PCR. Next, sequences encoding the GFP open reading frame 1997; Czaplinski et al. 1998). (ORF) without the termination codon were amplified from It is still unclear how nonsense mRNAs are initially plasmid pRSETB (courtesy of L. Robinson). Restriction sites identified as substrates for NMD. The Upf proteins are were engineered into the PCR product to allow insertion immediately downstream of the UPF3 promoter. Finally, the UPF3 not associated with all translating ribosomes (Atkin et ORF and sequences corresponding to the UPF3 3 UTR were al. 1995; Maderazo et al. 2000). Upf1p is at least 100amplified via PCR using pLS17 as a template. Using unique fold less abundant than ribosomes. Furthermore, Upf2p restriction sites, the PCR product was subcloned downstream and Upf3p are 10to 20-fold less abundant than Upf1p. of the GFP ORF. The entire cassette was cloned into pRS316 The low abundance of the Upf proteins suggests the and pRS426, creating pNE36 and pNE39, respectively. The plasmid expressing Upf3p-Triple-GFP was constructed by existence of a mechanism that serves to specifically rerecombinational cloning. pNE36 was digested with HindIII cruit the Upf proteins to the site of premature translaand BseRI. Sequences encoding the upf3-Triple ORF were tion termination of nonsense mRNAs. PCR amplifed from template pRLS125 and transformed into We showed previously that Upf3p contains a funcstrain RSy5 along with linearized pNE36. Recombined plastional nuclear export sequence, suggesting that Upf3p mids were rescued and sequenced. The upf3-Triple-GFP cassette was subcloned into pRS426, creating pAF14. To create may function in an early step in nonsense mRNA recruitpAF51, upf3-Triple-REV-GFP, the BseRI-KpnI fragment of ment (Shirley et al. 1998). However, it was not known pRLS141 was purified and subcloned into the same sites of at that time whether nuclear entry and exit is required pAF14. The construction of additional plasmids is described for NMD. Nuclear localization was recently demonbelow or was described previously (Shirley et al. 1998). strated for the human homologs of Upf3p. Several isoTwo-hybrid assay: The two-hybrid vectors used were pGBDU-C1,C2 and pGBDU-C1,C2 (Table 1; James et al. 1996). forms of hUpf3p were shown to shuttle between the Most of the translational fusions between ORFs of interest nucleus and the cytoplasm in cell culture heterokaryons and the Gal4 activation domains (AD) or binding domains (Lykke-Andersen et al. 2000; Serin et al. 2001). In this (BD) were generally constructed as follows. Primers were dearticle, we demonstrate that the import of yeast Upf3p signed to amplify the entire ORF of interest from plasmid into the nucleus is mediated by the importin/ heterDNA containing the gene sequence. The primers were used to engineer unique restriction sites and the 5 and 3 ends odimer. We further demonstrate that the export of 1467 Nuclear Import and Export of Upf3p