Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.

Highlights d Disome profiling reveals widespread ribosome collisions in vertebrates d Ribosomes are in queues at Pro-Pro/Gly/Asp, Arg-X-Lys, stop codons, and 3 0 UTRs d The positively charged nascent chain weakens the eIF5Amediated rescue of disomes d The stalled disomes on XBP1u mRNA are an endogenous substrate of RQC

FUS is an RNA/DNA-binding protein involved in multiple steps of gene expression and is associated with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). However, the specific disease-causing and/or modifying mechanism mediated by FUS is largely unknown. Here we evaluate intrinsic roles of FUS on synaptic functions and animal behaviours. We find that FUS depletion downregulates GluA1, a subunit of AMPA receptor. FUS binds GluA1 mRNA in the vicinity of the 3′ terminus and controls poly (A) tail maintenance, thus regulating stability. GluA1 reduction upon FUS knockdown reduces miniature EPSC amplitude both in cultured neurons and in vivo. FUS knockdown in hippocampus attenuates dendritic spine maturation and causes behavioural aberrations including hyperactivity, disinhibition and social interaction defects, which are partly ameliorated by GluA1 reintroduction. These results highlight the pivotal role of FUS in regulating GluA1 mRNA stability, post-synaptic function and FTLD-like animal behaviours.

Summary Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity.

In eukaryotic translation initiation, the eIF2 . GTP/MettRNA iMet ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2 . GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Be catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA i Met , and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Be mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Be with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Be segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Be to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA i Met , suggesting that tRNA iMet is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.

Fragile X Syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of Fmr1, which encodes the translational repressor protein FMRP. FMRP and CPEB, an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs, and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1-/y Cpeb−/− double knockout mice displayed significant amelioration of biochemical, morphological, electrophysiological, and behavioral phenotypes associated with FXS. Acute depletion of CPEB in the hippocampus of Fmr1 -/y mice rescued working memory deficits, demonstrating reversal of this FXS phenotype in adults. Finally, we find that FMRP and CPEB balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS, and that the maintenance of this homeostasis by FMRP and CPEB is necessary for normal neurologic function.

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cellfree translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA⅐70 S⅐fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.Translation initiation plays a pivotal role in the translational control of gene expression. Although the details of the mechanism that initiates translation vary among eukaryotes, prokaryotes, and archae, the productivity of translation in general is largely dependent on the formation of the initiation complex, which involves the binding of the mRNA to the ribosome and the recognition of the initiation codon. In eubacteria, translation initiation involves three steps. First, the 70 S ribosome is induced to dissociate into the 30 S and 50 S subunits by initiation factor (IF) 1 3 and IF1. Second, mRNA and the fMettRNA⅐IF2 complex bind the 30 S subunit in a random order (1), which results in the formation of the 30 S initiation complex. Finally, the 50 S subunit joins the 30 S initiation complex, thereby forming the 70 S initiation complex that is now set for the transition into the elongation phase (for review, see Refs. 2 and 3). As indicated, three IFs, namely, IF1, IF2, and IF3, modulate the kinetics of these processes. IF2 conveys fMettRNA onto the ribosomal P site (2, 4) and promotes the subsequent association of the 30 S and 50 S subunits (5). IF3 acts as a dissociation and fidelity factor that binds the 30 S subunit to prevent it from rejoining the 50 S subunit and ensures the proper positioning of the start codon and the initiator tRNA in the P site (4, 6, 7). IF1 promotes the activities of IF2 and IF3 (2) and protects the A site together with IF2 during the initiation event (8, 9).The processes described above have been studied intensively using canonical mRNAs that bear 5Ј-leader sequences containing a ribosome binding site such as the Shine-Dalgarno (SD) sequence (10). However, mRNAs that lack 5Ј-leader sequences do ...

miRNAs are small non-coding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The non-canonical polymerase Gld2 has been implicated in the stabilization of miR-122 possibly by catalyzing 3′ monoadenylation, however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2 involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3′ end. These results establish a novel mechanism of miRNA stability and resulting post-transcriptional gene regulation.