Mechanistic insights into tRNA translocation on the ribosome

The ribosome is a highly conserved cellular complex and constitutes the center of protein biosynthesis. As the ribosome consists to about 2/3 of ribosomal RNA (rRNA), the rRNA is involved in most steps of translation. In order to investigate the role of some defined rRNA residues in different aspects of translation we use the atomic mutagenesis approach. This method allows the site-specific incorporation of unnatural nucleosides into the rRNA in the context of the complete 70S from Thermus aquaticus, and thereby exceeds the possibilities of conventional mutagenesis. We first studied ribosome-stimulated EF-G GTP hydrolysis. Here, we could show that the non-bridging phosphate oxygen of A2662, which is part of the Sarcin-Ricin-Loop, is required for EF-G GTPase activation by the ribosome. EF-G GTPase is a crucial step for tRNA translocation from the A- to the P-site, and from the P- to the E-site, respectively. We furthermore used the atomic mutagenesis approach to more precisely characterize the 23S rRNA functional groups involved in E-site tRNA binding. While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [1].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

The aminoacyl-tRNA synthetases of Drosophila melanogaster

Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field.

Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition

Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.

Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNA(Ile). The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNA(Ile) precursor 3' end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNA(Ile) displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNA(Ile) precursor 3' end processing and contribute to the molecular pathomechanism of this mutation.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Acute respiratory distress syndrome secondary to antisynthetase syndrome is reversible with tacrolimus

Polymyositis and interstitial lung diseases, predominantly nonspecific interstitial pneumonia (NSIP), are known to be frequent in antisynthetase syndrome, where anti-aminoacyl-tRNA synthetase antibodies are often identified. An unusual case of acute respiratory distress syndrome, secondary to such proven NSIP of cellular type with predominant CD8 lymphocytes, is described herein. The patient described in the present case study initially had a poor recovery with high dose of steroids, but this was followed by a good improvement after the prescription of tacrolimus and a low dose of prednisone. A precise diagnosis in similar circumstances may be life-saving, allowing the successful application of new immunosuppressants.

The Single Mitochondrial Porin of Trypanosoma brucei is the Main Metabolite Transporter in the Outer Mitochondrial Membrane

All mitochondria have integral outer membrane proteins with beta-barrel structures including the conserved metabolite transporter VDAC (voltage dependent anion channel) and the conserved protein import channel Tom40. Bioinformatic searches of the Trypanosoma brucei genome for either VDAC or Tom40 identified a single open reading frame, with sequence analysis suggesting that VDACs and Tom40s are ancestrally related and should be grouped into the same protein family: the mitochondrial porins. The single T. brucei mitochondrial porin is essential only under growth conditions that depend on oxidative phosphorylation. Mitochondria isolated from homozygous knockout cells did not produce adenosine-triphosphate (ATP) in response to added substrates, but ATP production was restored by physical disruption of the outer membrane. These results demonstrate that the mitochondrial porin identified in T. brucei is the main metabolite channel in the outer membrane and therefore the functional orthologue of VDAC. No distinct Tom40 was identified in T. brucei. In addition to mitochondrial proteins, T. brucei imports all mitochondrial tRNAs from the cytosol. Isolated mitochondria from the VDAC knockout cells import tRNA as efficiently as wild-type. Thus, unlike the scenario in plants, VDAC is not required for mitochondrial tRNA import in T. brucei.

Magnetic resonance imaging and genetic investigation of a case of Rottweiler leukoencephalomyelopathy.

BACKGROUND Leukoencephalomyelopathy is an inherited neurodegenerative disorder that affects the white matter of the spinal cord and brain and is known to occur in the Rottweiler breed. Due to the lack of a genetic test for this disorder, post mortem neuropathological examinations are required to confirm the diagnosis. Leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate levels is a rare, autosomal recessive disorder in humans that was recently described to have clinical features and magnetic resonance imaging (MRI) findings that are similar to the histopathologic lesions that define leukoencephalomyelopathy in Rottweilers. Leukoencephalopathy with brain stem and spinal cord involvement is caused by mutations in the DARS2 gene, which encodes a mitochondrial aspartyl-tRNA synthetase. The objective of this case report is to present the results of MRI and candidate gene analysis of a case of Rottweiler leukoencephalomyelopathy to investigate the hypothesis that leukoencephalomyelopathy in Rottweilers could serve as an animal model of human leukoencephalopathy with brain stem and spinal cord involvement. CASE PRESENTATION A two-and-a-half-year-old male purebred Rottweiler was evaluated for generalised progressive ataxia with hypermetria that was most evident in the thoracic limbs. MRI (T2-weighted) demonstrated well-circumscribed hyperintense signals within both lateral funiculi that extended from the level of the first to the sixth cervical vertebral body. A neurodegenerative disorder was suspected based on the progressive clinical course and MRI findings, and Rottweiler leukoencephalomyelopathy was subsequently confirmed via histopathology. The DARS2 gene was investigated as a causative candidate, but a sequence analysis failed to identify any disease-associated variants in the DNA sequence. CONCLUSION It was concluded that MRI may aid in the pre-mortem diagnosis of suspected cases of leukoencephalomyelopathy. Genes other than DARS2 may be involved in Rottweiler leukoencephalomyelopathy and may also be relevant in human leukoencephalopathy with brain stem and spinal cord involvement.

An mRNA-Derived Noncoding RNA Targets and Regulates the Ribosome.

The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.