A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence.
In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd- phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd-. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd- phenotype even when export is restored.
Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison, WI, USA.
R L
RL
Shirley
M J
MJ
Lelivelt
L R
LR
Schenkman
J N
JN
Dahlseid
M R
MR
Culbertson
D019943:Amino Acid Substitution; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D017930:Genes, Reporter; D015757:Genes, rev; D015497:HIV-1 / Q000235:genetics; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C096360:UPF3 protein, S cerevisiae