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.

The cancer drug rocaglamide A cements the RNA helicase eIF4A on polypurine sequences and thereby prevents scanning of the 43S subunit along the messenger RNA, highlighting how a drug can act by stabilizing sequence-selective RNA–protein interactions. The cancer drug rocaglamide A (RocA) inhibits translation of a subclass of RNA transcripts. Because it targets the RNA helicase eIF4A, the specificity of RocA was thought to reside in the highly structured 5′ untranslated region of messenger RNAs that require eIF4A activity. Nicholas Ingolia and colleagues now show that the mechanism of inhibition has a different basis. RocA cements eIF4A on polypurine sequences and thereby prevents scanning of the 43S subunit along the messenger RNA. This report highlights how a drug can act by stabilizing sequence-selective RNA–protein interactions. Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs1,2,3,4. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5′ untranslated regions 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′ untranslated regions is only a minor determinant for RocA selectivity and that 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.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

This study shows that a vertebrate-specific ubiquitin ligase modulates neural crest specification in Xenopus development and human embryonic stem-cell differentiation; a proteomics approach reveals that the CUL3KBTBD8 ligase modulates translation by targeting the modulators of ribosomes production NOLC1 and its paralogue TCOF1, which is mutated in a neural-crest-associated syndrome. Changes in chromatin architecture and transcriptional programs direct cell differentiation during development, but little is known about the post-transcriptional regulation of cell fate determination. Michael Rape and colleagues show that a vertebrate-specific ubiquitin ligase modulates neural crest specification in the developing Xenopus embryo and in mouse embryonic stem cell differentiation. Using a proteomics approach, they find that the CUL3KBTBD8 ligase modulates translation by targeting the modulators of ribosome production, NOLC1 and its paralogue TCOF1. The TCOF1 locus is mutated in the neural crest associated condition known as Treacher Collins syndrome. Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3KBTBD8) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome2,3. Ubiquitylation drives formation of a TCOF1–NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.