An unresolved issue is how many different modes of decapp- ing exist in yeast cells and therefore how the diversity of mRNA decay rates is achieved. Decapping can also be negatively regulated on speciﬁc mRNAs. For example, the Khd1 protein binds to and limits the decapping on the Mtl1 mRNA (Mauchi et al. 2010). Since Khd1 can bind eIF4G and inhibit translation (Paquin et al. 2007), this may allow for the formation of a mRNP complex that is limited for translation, but protected from decapping. Since Khd1 can affect mRNA localization (Hasegawa et al. 2008), such a mechanism may be impor- tant in keeping mRNAs in a stable, but untranslating, state while the mRNAs localize to speciﬁc regions of the cell. This may be a common mechanism of mRNA control since mul- tiple translation repressors that can affect decapping also bind directly to eIF4G and can repress translation (Rajyaguru et al. 2012). Similarly, the Pub1 protein binds to, and stabil- izes, a signiﬁcant subset of yeast mRNAs, presumably by inhibiting decapping, although this has not been directly demonstrated (Ruiz-Echevarria and Peltz 2000; Duttagupta et al. 2005). mRNA decapping and P-bodies: The mRNA degradation machinery can also be concentrated in speciﬁc cytoplasmic mRNP aggregates referred to as P-bodies (Sheth and Parker 2003; see Figure 2). P-bodies are aggregates of untranslat- ing mRNAs that are associated with the mRNA decapping machinery, and to a lesser extent, with the deadenylases (Parker and Sheth 2007). P-bodies are proportional to the pool of untranslating mRNA associated with the decapping machinery (Teixeira et al. 2005). Consistent with this view, if mRNAs are trapped in polysomes, P-bodies decrease, whereas decreases in translation initiation increase the pool of mRNAs in P-bodies (Teixeira et al. 2005). Moreover, when mRNA decay is limited at the actual step of decapping or 59 to 39 degradation, P-bodies increase (Sheth and Parker 2003; Teixeira and Parker 2007). mRNAs within P-bodies can return to translation (Brengues et al. 2005), and this may occur through the transition of mRNAs from P-bodies to stress granules, which are aggregates of untranslating mRNAs assocatied with translation initation factors and RNA-binding proteins (reviewed in Buchan and Parker 2009). Note that the putative transfer of an mRNA from a P-body to a stress granule would correspond to an ex- change of the mRNA decapping machinery for translation initiation factors (Buchan et al. 2008). This constitutes an “mRNA cycle” wherein mRNAs can exit translation either for degradation or to eventually return (Figure 2), which may play a role in the regulation of both translation and mRNA degradation (Balagopal and Parker 2009). Interestingly, re- cent genomic analyses have identiﬁed at least 400 different mRNAs, compromising a large percentage of the transcrip- tome, that can recycle from repression to translation upon stress relief (Arribere et al. 2011). An unresolved issue is the role of P-body formation per se in the decapping of mRNAs. The aggregation of individual mRNPs into larger P-bodies is largely dependent on the YjeF domain of the Edc3 protein (Decker et al. 2007), although P-bodies can still form to some extent on the basis of a “prion” domain on Lsm4 (Decker et al. 2007; Reijns et al. 2008) and some Pat1-dependent aggregation (Buchan et al. 2008). How these aggregation motifs affect mRNA decay rates is unclear. In one report, strains lacking Edc3 and/or the aggregation domain of Lsm4 did not show any changes in the decay rates of the MFA2 mRNA (Decker et al. 2007), although in another report a strain lacking the Lsm4 aggre- gation motif did show a modest change in MFA2 mRNA decay rates (Reijns et al. 2008). The current simplest inter- pretation is that the formation of large P-body aggregates is not required for mRNA decapping but might affect the rate of decapping either in certain conditions or for subsets of mRNAs. Relationship of decapping to ongoing translation elonga- tion: An unresolved issue is how ongoing translation elongation affects the decapping of yeast mRNAs. Some evidence argues that decapping can be inhibited by elongat- ing ribosomes. First, inhibition of translation elongation by chemicals such as cycloheximide or sodarin leads to decreases in the rate of decapping (Beelman and Parker 1994; Cereghino et al. 1995; S. Jain and R. Parker, unpublished observation). However, this could also be due to indirect effects since cyclo- heximide can stabilize mRNAs that are never translated as well (Beelman and Parker 1994). Second, the rate of decapping of NMD substrates is proportional to the length of the ORF (Cao and Parker 2003). Moreover, in some cases, shortening the length of the ORF can lead to faster decay of speciﬁc mRNAs, although this could be due to loss of speciﬁc stabilizing sequences (Heaton et al. 1992). These results raise the possi- bility that mRNAs harboring elongating ribosomes have slower rates of decapping than ribosome-free mRNAs. In contrast, several observations argue that decapping can occur while ribosomes are still associated with mRNAs. First, in the presence of cycloheximide mRNAs are seen to be shortened from their 59 ends and degraded in a 59 to 39 manner to internal sites that are thought to be stalled ribo- somes, although this rate is slower than normal rates of decapping (Beelman and Parker 1994; Cereghino et al. 1995). Similarly, mRNAs with strong stalls in translation elongation have been argued to generate mRNA fragments by decapping and 59 to 39 degradation to the stalled ribo- somes (Hu et al. 2009), but more recent studies argue that these mRNA fragments may be produced by an endonucle- ase cleavage triggered by stalled ribosomes, referred to as no-go decay (NGD; see below) (D. Muhlrad and R. Parker, unpublished data). Additional evidence for decapping occur- ring on mRNAs engaged in elongation is that decapped mRNAs, either in wild-type cells or in xrn1Δ strains, appear to associate with polysomes, suggesting they are decapped while bound to elongating ribosomes (Hu et al. 2009). Taken together, the best current interpretation is that decapping can occur while mRNAs are associated with ribo- somes, but that ribosomes may also play a role in limiting the rates of decapping. RNA Decay in Yeast 681