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FTSJ1 is a conserved human 2′-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novelFTSJ1pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carryingFTSJ1mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed inDrosophilaand are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.

tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs with distinct cellular functions. Here, we studied the contribution of tRFs to the regulation of postnatal β cell maturation, a critical process that may lead to diabetes susceptibility in adulthood. We identified three tRFs abundant in neonatal rat islets originating from 5' halves (tiRNA-5s) of histidine and glutamate tRNAs. Their inhibition in these islets reduced β cell proliferation and insulin secretion. Mitochondrial respiration was also perturbed, fitting with the mitochondrial enrichment of nuclear-encoded tiRNA-5 HisGTG and tiRNA-5 GluCTC . Notably, tiRNA-5 inhibition reduced Mpc1, a mitochondrial pyruvate carrier whose knock down largely phenocopied tiRNA-5 inhibition. tiRNA-5 HisGTG interactome revealed binding to Musashi-1, which was essential for the mitochondrial enrichment of tiRNA-5 HisGTG . Finally, tiRNA-5s were dysregulated in the islets of diabetic and diabetes-prone animals. Altogether, tiRNA-5s represent a class of regulators of β cell maturation, and their deregulation in neonatal islets may lead to diabetes susceptibility in adulthood.

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.

2′-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

N6-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t6A) is a universal modification essential for translational accuracy and efficiency. The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex., The biosynthesis of N6-threonylcarbamoylated adenosine 37 in tRNA (t6A) involves the YRDC enzyme and the KEOPS complex. Here, the authors report mutations in YRDC and the KEOPS component GON7 in Galloway-Mowat syndrome and determine the crystal structure of a GON7-containg subcomplex that suggests a role in KEOPS complex stability.

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that only ∼40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.

We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large-scale deletions of mtDNA. Findings were compared with those obtained from asymptomatic relatives with the 3243A>G mutation, myotonic dystrophy patients, and healthy subjects. Plasma lactate concentration, maximal oxygen uptake, and ragged-red fibers/cytochrome c-negative fibers in muscle were also determined. Only 10% of patients with the 3243A>G point mutation had decreased enzyme activity of one or more RC complexes, whereas this was the case for 83% of patients with other point mutations and 62% of patients with deletions. Abnormal muscle histochemistry was found in 65%, 100%, and 85% of patients, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in milk samples from cows with and without mastitis. Elucidating their relevance in bovine udder health is hampered because identification at the species level, if done at all, used to be performed based on phenotypic features. To provide a rapid, cheap, and easy-to-use genotypic technique that can be used to identify CNS species from milk and teat apices from cows, the performance of transfer RNA-intergenic spacer PCR (tDNA-PCR) in combination with capillary electrophoresis was evaluated. After updating the tDNA library with CNS reference strains, 288 field isolates were identified with tDNA-PCR and gene sequencing, and the latter was used as the reference method. The field isolates were divided in 2 groups of 144. Isolates of the first group were identified with tDNA-PCR with a typeability of 81.9% and an accuracy of 94.1%. Peak patterns of these isolates were then added to the tDNA library with species identity as determined by DNA sequencing. The second group was identified with the updated tDNA library, resulting in 91.0% typeability and 99.2% accuracy. This study showed that the updated tDNA-PCR in combination with capillary electrophoresis was almost as accurate as gene sequencing but faster and cheaper (only $3 per isolate), and is a useful tool in observational studies concerning the epidemiology of bovine CNS species.

Background Defects in the oxidative phosphorylation system can cause a broad spectrum of clinical symptoms ranging from an isolated myopathy to a multisystemic disorder. Objective To study and identify the underlying molecular defect in a patient with limb-girdle myopathy. Design Biochemical, histochemical, and immunocytochemical analyses were performed in combination with polymerase chain reaction–single-strand conformation polymorphism and restriction fragment length polymorphism–polymerase chain reaction techniques. Setting University hospital. Patient A 48-year-old woman with limb-girdle myopathy. Main Outcome Measures The pathogenic characteristics of the identified nucleotide alterations were defined using single-muscle fiber analysis. Results A complex III deficiency was detected using blue native–polyacrylamide gel electrophoresis, while immunocytochemical results showed a mosaic staining pattern for complexes I and IV. After molecular analyses, 2 novel heteroplasmic mitochondrial DNA (mtDNA) nucleotide aberrations, m.5888insA and m.14639A>G, were identified in muscle tissue. Single-muscle fiber analyses demonstrated that cytochrome c oxidase–deficient fibers, compared with cytochrome c oxidase–positive fibers, harbored statistically significantly higher levels of both mtDNA mutations ( P t test). Conclusions These results, together with previously defined canonical criteria determining the pathogenic characteristics of mtDNA mutations, suggest that both nucleotide changes are pathogenic mutations. To our knowledge, this is only the third report of the coexistence of 2 pathogenic mtDNA mutations present in different genes within individual skeletal muscle fibers of a patient.

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.