independent decapping (Muhlrad and Parker 1994), slightly increased rates of 39 to 59 degradation after deadenylation (Cao and Parker 2003; Mitchell and Tollervey 2003), and translation repression (Muhlrad and Parker 1999b). Consis- tent with NMD targeting mRNAs for translation repression, when decapping or 59 to 39 degradation is blocked, NMD substrates accumulate as repressed mRNAs in P-bodies in an Upf1-dependent manner (Sheth and Parker 2006). NMD has been suggested to be coupled to degradation of the nascent peptide in an Upf1-dependent manner (Kuroha et al. 2009). In this manner, not only would the mRNA be degraded, but any potential dominant-negative peptides produced would also be rapidly destroyed. Interestingly, Upf1 has been suggested to have ubiquitin ligase activity, and mutations that affect this activity alter the process of NMD for RNA degradation (Takahashi et al. 2008), although the sites of these mutations would also be predicted to dis- rupt Upf1 interaction with Upf2, which is known to be re- quired for NMD (He et al. 1996, 1997; Clerici et al. 2009). However, how general NMD stimulated protein decay remains to be established since a peptide from a different NMD mRNA substrate shows the same decay rates in wild- type and upf1Δ cells (Muhlrad and Parker 1999b). Transcripts appear to be targeted for the diverse effects of NMD in two steps (Figure 3). Several observations suggest that, in an initial step, Upf1, which is a member of the SF1 protein superfamily of nucleic acid helicases (Fairman-Wil- liams et al. 2010), interacts with the translation termination complex and alters the nature of translation termination (reviewed in Baker and Parker 2004). The most direct evi- dence is that a ribosome toeprint generated at a normal translation termination codon is distinct from the toeprint of a ribosome terminating at a premature termination codon in a manner dependent on Upf1p (Amrani et al. 2004). In addition, the Upf1, -2, and -3 proteins co-immunoprecipitate with the eukaryotic translation termination factors eRF1 and/or eRF3 (Czaplinski et al. 1998; Wang et al. 2001). Although upf1Δ mutants can show increased rates of stop- codon readthrough, this effect appears to be due to stabili- zation and increased expression in the upf1Δ strain of a mag- nesium transporter, which increases intracellular Mg++ and leads to increased miscoding of stop codons (Johansson and Jacobson 2010). Interestingly, a defect in upf2Δ and upf3Δ strains in stop codon readthrough can be suppressed by overexpression of Upf1, suggesting that Upf1 directly affects translation, independently of Upf2 and Upf3, of mRNAs with some stop codons, perhaps by inhibiting translation initation (Muhlrad and Parker 1999b; Maderazo et al. 2000). Consis- tent with this view, Upf1 can associate with polysomes in- dependently of Upf2 and Upf3 (Atkin et al. 1997). The upf2Δ and upf3Δ strains may have decreased Upf1 function since in these strains Upf1 accumulates in P-bodies, and therefore the majority of Upf1 may not be available to affect trans- lation termination (Sheth and Parker 2006). Moreover, because NMD substrates accumulate in P-bodies indepen- dently of Upf2 and Upf3, the simplest model is that Upf1 684 R. Parker is sufﬁcient to repress translation of the mRNA (Sheth and Parker 2006). A second step in NMD appears to be the interaction of Upf2 and Upf3 with Upf1, and this triggers the degradation of the mRNA. This conclusion is supported by the Upf2 and Upf3 independent effects of Upf1 on translation read- through and targeting of NMD substrates to P-bodies (Maderazo et al. 2000; Sheth and Parker 2006). Upf3 is an RNA-binding protein that is proposed to load on mRNAs in the nucleus (Shirley et al. 2002). Upf2 interacts with both Upf3 and Upf1, and its binding to Upf1 reduces the Upf1–RNA interaction and enhances the helicase activity of Upf1 (Chakrabarti et al. 2011). This suggests that, follow- ing translation termination altered by Upf1, interaction of Upf2 with Upf1 would enhance Upf1 catalytic properties and lead to mRNP rearrangements that trigger mRNA deg- radation, possibly by rearrangements of the mRNP or by altering the fate of the terminating ribosome (Ghosh et al. 2010). Consistent with that model, mutations inactivating the ATPase activity of Upf1 also accumulate mRNAs in P-bodies (Sheth and Parker 2006). A key issue is how the speciﬁcity of NMD is determined. In principle, an mRNA will be targeted for NMD on the basis of the nature of translation termination (is it “aberrant” or not?) and whether Upf2/Upf3 can inﬂuence Upf1 after al- tered termination. Thus, the speciﬁcity of NMD is deter- mined by factors that inﬂuence translation termination and whether Upf2/Upf3 is associated with the mRNA after termination. One factor that contributes to proper transla- tion termination is Pab1, which is known to interact with the translation termination factors, and, when tethered to the mRNA near a premature stop codon, can prevent NMD on that mRNA (Amrani et al. 2004). However, strains lacking Pab1, as well as poly(A)-mRNAs, still show Upf1-dependent degradation of mRNAs with premature stop codons, indicat- ing that additional factors also contribute to NMD targeting, although whether those factors inﬂuence Upf1 effects on termination or effect a downstream step in NMD is not re- solved (Caponigro and Parker 1995; Meaux et al. 2008). Thus, a key issue is determining what other features of an mRNA inﬂuence the nature of translation termination and Upf2/Upf3 interaction with the mRNA. Although currently controversial, one potential contribution is elements in cod- ing regions, sometimes referred to as downstream sequence elements (reviewed in Gonzalez et al. 2001), that might recruit Upf3 and Upf2 to mRNAs, and, if Upf2 and Upf3 are not removed by elongating ribosomes, might lead to triggering NMD after upstream termination. The multistep process of NMD in yeast is also revealed by the observation that 59 proximal stop codons trigger faster mRNA degradation than stop codons farther into the ORF (Losson and Lacroute 1979; Peltz et al. 1993; Cao and Parker 2003). Strikingly, as judged by Upf1-promoted dead- enylation, all premature stop codons are recognized as ab- errant, but the position of the stop codon simply dictates differences in the actual rates of Upf1-promoted decapping