In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.

In eukaryotes, shortening of the 3-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes-Caf1-Ccr4 and Pan2-Pan3-play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1-Ccr4 and Pan2-Pan3. Interestingly, translation termination complexes eRF1-eRF3, Pan2-Pan3, and Caf1-Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1-eRF3 for the deadenylase occurs on PABPC1. Consequently, PABPC1 binding leads to the activation of Pan2-Pan3 and Caf1-Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2-Pan3 and Caf1-Ccr4 in cooperation with eRF3 and PABPC1.[Keywords: Translation termination; deadenylation; eRF3; PABPC1] Supplemental material is available at http://www.genesdev.org.

No abstract

In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.

We study the influence of surface roughness on the adhesion between elastic solids. We present experimental data for the force necessary to pull off rubber balls from hard rough substrates. We show that the effective adhesion (or the pull-off force) can be calculated accurately from the surface roughness power spectra obtained from the measured surface height profile.

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3 end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

Using Ar beam etching in vacuum, strong bonding of Si wafers is attained at room temperature. With appropriate etching time, the bonding occurs spontaneously without any load to force two wafers together. However, surface roughness of the wafers increases during Ar beam etching. Because surface roughness has a strong influence on wafer bonding, long etching time degrades the bonding strength. Using atomic force microscope, we measured surface roughness enhancement caused by Ar beam etching, and investigated the relationship between surface roughness and bonding properties such as strength and interfacial voids. The results agree well with theoretical predictions using elastic theory and energy gain by bond formation. A guideline for successful room-temperature bonding is proposed from these results.

In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation.The 3Ј ends of almost all eukaryotic mRNAs and their precursors consist of homopolymeric tails of adenosine, or poly(A), that are added by poly(A) polymerase (PAP) during the process of 3Ј end formation. There are two classes of poly(A) binding proteins (PABPs) in mammalian cells (32,40). One class is exemplified by PABPN1, formerly called PABP2. PABPN1 is primarily nuclear and plays a role in the synthesis of poly(A) tails, but it also shuttles between the nucleus and cytoplasm (5,8,9,31,53). The other class consists of the primarily cytoplasmic PABPs, of which PABPC1, formerly called PABI or PABP1, is the major form in mammalian somatic cells (40). In humans, at least four separate PABPC genes and four pseudogenes have been identified (40). PABPC1 influences mRNA translation and decay (18,20,23,27,29,30,50,(54)(55)(56), and it shuttles between the nucleus and cytoplasm of at least some mammalian cells (1,57,58). Consistent with the preferential compartmentalization of PABPN1 to the nucleus and PABPC1 to the cytoplasm, a physical interaction has been detected between PAP and PABPN1 but not PABPC1 (28). Furthermore, PABPN1 associates with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore (2). Given that PABPC1 can exist within nuclei, a key issue is whether PABPC1 binds to transcripts inside the nucleus, and if it does, at which step in mRNA maturation.The 5Ј ends of eukaryotic mRNAs and their precursors are capped, and the cap is initially bound by the mostly nuclear cap binding protein (CBP) heterodimer of CBP80 and CBP20 (25, 35, 38), which will be called CBP80/CBP20. CBP80/CBP20 is detectably replaced by the mostly cytoplasmic eukaryotic translation initiation factor 4E (eIF4E) only after introns have been removed by splicing (35). Evidence indicates that PABPC1 is a component...