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.

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumor cells and repress translation of specific mRNAs1-4. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its mRNA selectivity is proposed to reflect highly structured 5′ UTRs that depend strongly on eIF4A-mediated unwinding5. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA1,2,6. Here, we show that secondary structure in 5′ UTRs is only a minor determinant for RocA selectivity and RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.

Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5–phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.