Relationship between Yeast Polyribosomes and Upf Proteins Required for Nonsense mRNA Decay

Abstract

In yeast, the accelerated rate of decay of nonsense mutant mRNAs, called nonsense-mediated mRNA decay, requires three proteins, Upf1p, Upf2p, and Upf3p. Single, double, and triple disruptions of the UPF genes had nearly identical effects on nonsense mRNA accumulation, suggesting that the encoded proteins function in a common pathway. We examined the distribution of epitope-tagged versions of Upf proteins by sucrose density gradient fractionation of soluble lysates and found that all three proteins co-distributed with 80 S ribosomal particles and polyribosomes. Treatment of lysates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into fractions containing 80 S ribosomal particles, as expected for proteins that are associated with polyribosomes. Mutations in the cysteine-rich (zinc finger) and RNA helicase domains of Upf1p caused loss of function, but the mutant proteins remained polyribosome-associated. Density gradient profiles for Upf1p were unchanged in the absence of Upf3p, and although similar, were modestly shifted to fractions lighter than those containing polyribosomes in the absence of Upf2p. Upf2p shifted toward heavier polyribosome fractions in the absence of Upf1p and into fractions containing 80 S particles and lighter fractions in the absence of Upf3p. Our results suggest that the association of Upf2p with polyribosomes typically found in a wild-type strain depends on the presence and opposing effects of Upf1p and Upf3p.The notion of a global pathway for eukaryotic mRNA decay suggested by early work in animal cells has recently been greatly advanced by studies in the yeast Saccharomyces cerevisiae (1-4). Using an in vivo transcriptional pulse, the temporal fate of newly synthesized mRNA was established by monitoring poly(A) tail length, loss of the m 7 Gppp cap, disappearance of the mRNA, and the appearance of degradation intermediates. mRNAs with shorter half-lives were generally subject to faster rates of deadenylation and decapping. Once the poly(A) tails were reduced to a short oligo(A) length (10 -12 nucleotides), the mRNAs were decapped and digested from the 5Ј end. Decapping requires Dcp1p (5). Processive degradation from the 5Ј end requires the product of the XRN1 gene, which is known to encode a 5Ј 3 3Ј exoribonuclease (6). Deadenylation-dependent decapping followed by 5Ј 3 3Ј exonucleolytic decay is likely to be the global default pathway for the degradation of most eukaryotic mRNAs.Yeast mRNAs containing a premature stop codon decay more rapidly than their wild-type counterparts (7). This accelerated decay, called nonsense-mediated mRNA decay (NMD), 1 requires cis-acting elements in the mRNA in addition to a premature stop codon (8). Premature translational termination triggers decapping at the 5Ј end of nonsense mRNAs with kinetics that are independent of deadenylation (9). Following decapping, decay proceeds through the Xrn1p-mediated nucleolysis that is common to intrinsic decay. These results support the view that when translation is prematurely terminat...

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

“…3D). These experiments showed that Upf1 copurified predominantly with 40S ribosomal subunits in upf2Δ, upf3Δ, and upf2Δupf3Δ strains, similar to the results observed in wild-type strains and consistent with earlier studies showing that polysome-association of Upf1 is independent of the other NMD factors (Atkin et al 1997). In several of the strains analyzed, a modest amount of Upf1 association with 60S subunits was observed; however, as monitored by Rps6 recovery, this appeared to reflect 40S subunit contamination in the 60S subunit preparations (Fig.…”

“…This conclusion is reinforced by experiments demonstrating a direct correlation between the extent of ribosome occupancy at a PTC and the degree to which NMD is activated (Gaba et al 2005). Additional links between translation and NMD include the observations that decapping and degradation of nonsense-containing transcripts occur while the transcript is associated with polyribosomes (Mangus and Jacobson 1999;Hu et al 2010) and that the Upf factors colocalize with polyribosomes, 80S ribosomes, and ribosomal subunits (Peltz et al 1993;Atkin et al 1995Atkin et al , 1997Mangus and Jacobson 1999;Ghosh et al 2010). Consistent with a dependence of NMD on translation termination, Upf1 interacts with the release factors eRF1 and eRF3 in humans and yeast, possibly to function in termination events using its helicase and RNA binding activities (Czaplinski et al 1998;Wang et al 2001;Ghosh et al 2010).…”

“…Interestingly, neither the C62Y nor the C84S mutations affect the association of Upf1 with polysomes or 40S ribosomal subunits ( Fig. 3B; Atkin et al 1997; S Ghosh and A Jacobson, unpubl. ), suggesting that Upf1:40S association may be a multivalent interaction involving multiple interacting epitopes.…”

Functional characterization of Upf1 targets inSchizosaccharomyces pombe

“…However, it is unclear whether some of the functions of Upf1 in fission yeast can be performed in the absence of Upf2 or Upf3, as described in other systems. 20,21,23 Addressing this question will require additional experiments.…”

“…There is also evidence that Upf1 and other components of NMD can play important functions in the cell independently of their role in the NMD process. [20][21][22][23][24] In fact, many targets of NMD factors do not have PTCs. 8,25 The fission yeast Schizosaccharomyces pombe contains Upf1 and Upf2 proteins, although the NMD mechanism is somehow different from mammals.…”