A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence

Abstract

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.

Ribosome Flow Model on a Ring

“…For example, in E. coli RNA degradation often begins with conversion of the 5 ′ -terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E [37]. In eukaryotes, such as Saccharomyces cerevisiae, intrinsic mRNA decay initiate with deadenylation that causes the shortening of the poly(A) tail at the 3 ′ end of the mRNA, followed by the removal of the cap at the 5 ′ end by the decapping enzyme, which leads to a rapid 5 ′ → 3 ′ degradation of the mRNA by an exoribonuclease [40]. In eukaryotes, the canonical scanning model requires free ends of the mRNA [21].…”

Interaction between Ski7p and Upf1p is required for nonsense-mediated 3'-to-5' mRNA decay in yeast

“…Loss-offunction mutations in proteins that are required for 3¢-to-5¢ mRNA degradation are synthetically lethal with lesions that block the other major decay pathway of decapping and 5¢-to-3¢ degradation (Jacobs Anderson and Parker, 1998). Therefore, we used a strain containing either dcp1-2 or rrp4-1 (Mitchell et al, 1996;Tharun and Parker, 1999). These are temperature-sensitive alleles and mRNA decay in these strains is severely inhibited at the restrictive temperature.…”

“…Upf2p rich in acidic amino acid residues interacts with both Upf1p and Upf3p (He et al ., 1997). Upf3p, which is a smaller protein highly rich in basic residues, contains nuclear import and export signals, and it has been shown to localize to the nucleus (Shirley et al ., 1998). Recognition of a termination codon as premature requires a cis ‐acting downstream element (DSE), which is located at the 3′ end of PTCs (Peltz et al ., 1993; Zhang et al ., 1995).…”

“…The exosome is a complex of 3¢-to-5¢ exonucleases consisting of 10 subunits (Mitchell et al, 1997;Allmang et al, 1999). In the cytoplasm, the exosome participates in mRNA turnover (Jacobs Anderson and Parker, 1998;van Hoof et al, 2000). The function of the cytoplasmic complex requires several cofactors, named Ski proteins.…”

The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts

“…The UPF3 gene encodes a smaller protein highly rich in basic residues (Lee and Culbertson, 1995). Upf3p has three sequence elements that resemble nuclear localization signals and two that resemble the leucine‐rich nuclear export signals (Shirley et al ., 1998). Cells harboring single or multiple deletions of the UPF genes demonstrate equivalent stabilization of the nonsense‐containing mRNAs, indicating that the Upf proteins either function as a complex or are components of one regulatory pathway (Cui et al ., 1995; He et al ., 1997).…”