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The tumorigenic mechanism for pancreatic ductal adenocarcinoma (PDAC) is not clear, although chronic inflammation is implicated. Here, we identified an inflammatory cytokine-regulated transfer RNA- derived (tRNA-derived) fragment, tRF-21VBY9PYKHD (tRF-21), as a tumor suppressor in PDAC progression. We found that the biogenesis of tRF-21 could be inhibited by leukemia inhibitory factor and IL-6 via the splicing factor SRSF5. Reduced tRF- 21 promoted AKT2/1-mediated heterogeneous nuclear ribonucleoprotein L (hnRNP L) phosphorylation, enhancing hnRNP L to interact with dead-box helicase 17 (DDX17) to form an alternative splicing complex. The provoked hnRNP L-DDX17 activity preferentially spliced Caspase 9 and mH2A1 pre-mRNAs to form Caspase9b and mH2A1.2, promoting PDAC cell malignant phenotypes. The tRF-21 levels were significantly lower in PDACs than in normal tissues, and patients with low tRF-21 levels had a poor prognosis. Treatment of mouse PDAC xenografts or patient-derived xenografts (PDXs) with tRF-21 mimics repressed tumor growth and metastasis. These results demonstrate that tRF-21 has a tumor-suppressive effect and is a potential therapeutic agent for PDAC., Introduction Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy, with a 5-year survival rate of less than 9% (1, 2). Most PDAC tumors are already in an advanced stage [...]

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars.sup.sti mutant mice results in an increase in the production of serine-mischarged tRNA.sup.Ala and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars.sup.sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNA.sup.Ala and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aars.sup.sti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.ANKRD16 attenuates neurodegeneration induced by a mutation in the editing domain of alanyl tRNA synthetase by directly accepting mis-activated serine from the synthetase before transfer to the tRNA, establishing a new mechanism by which editing defects are prevented., Author(s): My-Nuong Vo [sup.1] , Markus Terrey [sup.2] [sup.3] [sup.4] [sup.5] , Jeong Woong Lee [sup.5] [sup.12] , Bappaditya Roy [sup.6] [sup.7] , James J. Moresco [sup.8] [sup.13] , Litao [...]

Antimalarial drugs have thus far been chiefly derived from two sourcesnatural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets., Author(s): Nobutaka Kato [1]; Eamon Comer [1]; Tomoyo Sakata-Kato [2]; Arvind Sharma [3]; Manmohan Sharma [3]; Micah Maetani [1, 4]; Jessica Bastien [1]; Nicolas M. Brancucci [2]; Joshua A. Bittker [...]

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system.sup.1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules.sup.1. Using a mouse model with a heteroplasmic mtDNA mutation.sup.2, we tested whether mitochondrial-targeted TALENs (mitoTALENs).sup.3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNA.sup.Ala levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice. Mitochondrial-targeted TALENs reduce mutation burden and correct biochemical defects in a mouse model of mitochondrial disease., Author(s): Sandra R. Bacman [sup.1] , Johanna H. K. Kauppila [sup.2] , Claudia V. Pereira [sup.1] , Nadee Nissanka [sup.1] , Maria Miranda [sup.2] , Milena Pinto [sup.1] , Sion [...]

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development., In all living cells, ribosomes are large ribonucleoprotein assemblies responsible for the accurate conversion of the genetic information encoded within mRNA into a corresponding protein. Although core functions of the [...]

Murine leukemia viruses (MLVs) and related retroelements are potently restricted in embryonic cells by postintegration transcriptional silencing, likely to protect the germ line from insertional mutagenesis. This silencing is in large part attributable to the presence of a nuclear repression complex, which targets a sequence element of the proviral DNA, the repressor-binding site. The repressor-binding site closely overlaps the tRNA primer binding site, a highly conserved sequence essential for virus replication and defining the site of initiation of DNA synthesis during reverse transcription. We have recently demonstrated that the cellular corepressor TRIM28 is recruited to the proline tRNA primer-binding site used by many MLVs and is required to mediate this silencing. Here, we show that TRIM28 is also required for the restriction of retroviruses using a completely distinct tRNA for the priming of their DNA synthesis, namely Lys-1,2 tRNA. These results generalize the role of TRIM28 in retroviral restriction and suggest that this system has evolved to restrict multiple retroviruses. HP1 | KAP-1 I MLV | PBS

Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Because the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we use a variety of known mutations in hmt-[tRNA.sup.Phe] to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-[tRNA.sup.Phe] mutations on aminoacylation, structure, and translation elongation-factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure, and elongation-factor binding. One notable exception was the G34A anticodon mutation of hmt-[tRNA.sup.Phe] (mitochondrial DNA mutation G611A), which is associated with MERRF (myoclonic epilepsy with ragged red fibers). In vitro, the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-[tRNA.sup.Phe] does not undergo adenosine-to-inosine (A-to-I) editing, ruling out miscoding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-[tRNA.sup.Phe] with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene. aminoacyl-tRNA synthetase | translation | mitochondria