Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors

Abstract

In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upfD strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1D extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1D extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.

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

“…Here, we address the Upf1 localization problem by providing biochemical and genetic evidence for the association of Upf1 with ribosomal subunits in the budding yeast Saccharomyces cerevisiae. First, we utilized independent approaches to confirm our earlier observations of Upf1 association with the 40S ribosomal subunit (Ghosh et al 2010). Having confirmed such interactions, we then defined the domains of Upf1 on which they depend as well as a specific ribosomal protein with which Upf1 interacts.…”

“…In yeast, ATPase-deficient Upf1 accumulates with NMD substrates in cytoplasmic processing bodies (Sheth and Parker 2006); and in human cells, ATPase-or helicase-deficient Upf1 leads to the accumulation of partially degraded 3 ′ decay intermediates (Franks et al 2010). These reports and others (Ghosh et al 2010) suggest a role for Upf1 in disassembling post-termination mRNPs in a mechanism that requires its ATPase and helicase activities. Notably, the maximal activation of these activities is stimulated by a complex of Upf2 and Upf3 (Chamieh et al 2008;Chakrabarti et al 2011).…”

“…Our previous experiments demonstrated that yeast Upf1 associates with purified 40S ribosomal subunits (Ghosh et al 2010). We have now explored this interaction further by testing whether Upf1 is retained by an alternative method of ribosome isolation, namely affinity purification.…”

“…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).…”

“…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). In vertebrates, Upf1 and its regulator Smg-1 interact with the release factors forming the SURF (Smg-1-Upf1-Release factors) complex, and this SURF-associated Upf1 appears to be able to interact with Upf2-Upf3 bound to the exon junction complex (Yamashita et al 2009;Kashima et al 2010).…”