tRNA-derived small RNA changes in bone marrow stem cells under hypoxia and osteogenic conduction.

BACKGROUND: Tissue engineering using bone mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic for bone regeneration. However, the effect of bone regeneration remains unsatisfactory due to the BMSCs' functional abnormality influenced by hypoxia. In this study, we attempt to explore the mechanism of osteogenic differentiation of BMSCs under hypoxic conditions from the perspective of non-coding RNA regulation. METHODS: The study employed BMSCs obtained from healthy donors and simulated hypoxia using CoCl2 stimulation. High-throughput sequencing technique was used to identify differential expression profiles of tRNA-derived small RNA (tsRNA) in three experimental groups: BMSCs-0d, BMSCs-7d and BMSCs-0d-CoCl2 . TargetScan and miRanda algorithms were used to determine tsRNA target genes, while Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were employed for the prediction of biological functions. Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR) was carried out on four selected differentially expressed tsRNAs. RESULTS: After the osteogenic induction and CoCl2 stimulated separately, there were 19 tsRNAs differentially expressed in BMSCs, including 14 upregulated and five downregulated. According to the analysis of biological information, these tsRNAs may regulate 311 potential target genes and mainly enrich the pathways such as metabolic pathways, Wnt signalling pathway, osteoclast differentiation, cellular senescence and mTOR signalling pathway. The results of Real-time RT-PCR for 3'tiRNA-41-GlnTTG-6, 3'tiRNA-42-LysTTT-8, 5'tiRNA-35-CysACA-1 and tRF3a-AsnGTT-9 were consistent with small RNA sequencing data. CONCLUSION: We discovered the tsRNA that changes the process of osteogenesis and hypoxia, which provides new targets for promoting survival and regeneration functions after BMSCs transplantation.

Ascophyllum nodosum polysaccharide regulates gut microbiota metabolites to protect against colonic inflammation in mice.

Ascophyllum nodosum polysaccharide (ANP) can protect against colonic inflammation but the underlying mechanism is still unclear. This study has determined the metabolites of gut microbiota regulated by ANP to reveal the mechanism of the anti-inflammation effect of ANP. Using an in vitro colonic fermentation model, the results indicate that gut microbiota could utilize a proportion of ANP to increase the concentrations of short-chain fatty acids (SCFAs) and decrease ammonia content. Metabolomics revealed that 46 differential metabolites, such as betaine, L-carnitine, and aminoimidazole carboxamide ribonucleotide (AICAR), could be altered by ANP. Metabolic pathway analysis showed that ANP mainly up-regulated the phenylalanine, tyrosine, and tryptophan biosynthesis and aminoacyl-tRNA biosynthesis, which were negatively correlated with inflammation progression. Interestingly, these metabolites associated with inflammation were also up-regulated by ANP in colitis mice, including betaine, L-carnitine, AICAR, N-acetyl-glutamine, tryptophan, and valine, which were mainly associated with amino acid metabolism and aminoacyl-tRNA biosynthesis. Furthermore, the metabolites modulated by ANP were associated with the relative abundances of Akkermansia, Bacteroides, Blautia, Coprobacillus, Enterobacter, and Klebsiella. Additionally, based on VIP values, betaine is a key metabolite after the ANP supplement in vitro and in vivo. As indicated by these findings, ANP can up-regulate the production of SCFAs, betaine, L-carnitine, and AICAR and aminoacyl-tRNA biosynthesis to protect against colonic inflammation and maintain intestinal health.

Tavaborole-Induced Inhibition of the Aminoacyl-tRNA Biosynthesis Pathway against Botrytis cinerea Contributes to Disease Control and Fruit Quality Preservation.

The inhibitory effect of tavaborole on the invasion of Botrytis cinerea in grapes and tomatoes, as well as the potential mechanism involved, was discovered in this study. Our findings showed that tavaborole inhibited Botrytis cinerea spore germination and mycelial expansion in vitro and that the control efficiency in vivo on fruit decay was dose-dependent, which was effective in reducing disease severity and maintaining the organoleptic quality of the fruit, such as reducing weight loss and retaining fruit hardness and titratable acid contents during storage. Furthermore, the precise mechanism of action was investigated further. Propidium iodide staining revealed that Botrytis cinerea treated with tavaborole lost membrane integrity. For further validation, cytoplasmic malondialdehyde accumulation and leakage of cytoplasmic constituents were determined. Notably, the inhibitory effect was also dependent on inhibiting the activities of aminoacyl-tRNA synthetases involved in the aminoacyl-tRNA biosynthesis pathway in Botrytis cinerea. The above findings concluded that tavaborole was effective against Botrytis cinerea infection in postharvest fruit, and a related mechanism was also discussed, which may provide references for the drug repurposing of tavaborole as a postharvest fungicide.

Phenotype microarray analysis reveals the biotransformation of Fusarium oxysporum f.sp. lycopersici influenced by Bacillus subtilis PBE-8 metabolites.

Herein, Bacillus subtilis PBE-8's biocontrol efficacy was evaluated through physiological and metabolic approaches against Fusarium oxysporum f.sp. lycopersici (FOL). The study elaborates on PBE-8's cell-free filtrate (CFF) antifungal activity through mycelial growth inhibition, metabolite profiling, and substrates utilization patterns. Additionally, under different CFF concentrations, reduction in spore count (94%-55%), biomass (50%), and cytoplasmic bulbous protrusions in mycelia were also observed. Furthermore, the effect of bacterial CFF on FOL metabolism was confirmed through GC-MS. CFF suppresses the concentration of aliphatic amino acids like L-valine, L-leucine, L-Isoleucine, glycine, and fatty acids such as linoleic acid and α- linolenic acid during the co-culturing conditions, which are essential for pathogenicity and resistance against host's systemic acquired resistance. The phenotype microarray assay revealed that CFF-treated FOL shows phenotype loss in 507 (56.58%) out of 896 substrates. Among 507, twenty-seven substrates showed significant phenotype loss, among which four substrates such as L-glutamic acid, L-glutamine, ammonia, and L-arginine are common in different crucial metabolic pathways of FOL, like alanine, aspartate, and glutamate metabolism, arginine and proline, carbon metabolism, arginine biosynthesis, nitrogen metabolism, amino-acyl tRNA synthesis, and biosynthesis of amino acids. The results suggest that PBE-8 CFF has certain antifungal metabolites that hinder the fungal metabolic pathways.

Effects of polystyrene microplastics acute exposure in the liver of swordtail fish (Xiphophorus helleri) revealed by LC-MS metabolomics.

As global pollution, microplastics pollution has aroused growing concerns. In our experiment, the effect of microplastics acute exposure on the liver of swordtail fish was investigated by using LC-MS metabolomics. Fishes treated with high concentration polystyrene microspheres (1 µm) for 72 h were divided into three concentration groups: (A) no microplastics, (B): 1 × 106 microspheres L-1, (C): 1 × 107 microspheres L-1. Metabolomic analysis indicated that exposure to microplastics caused alterations of metabolic profiles in swordtail fish, including 37 differential metabolites were identified in B vs. A, screened out ten significant metabolites, which involved 14 metabolic pathways. One hundred three differential metabolites were identified in C vs. A, screened out 16 significant metabolites, which involved 30 metabolic pathways. Six significant metabolites were overlapping in group B vs. A and C vs. A; they are 3-hydroxyanthranilic acid, l-histidine, citrulline, linoleic acid, pantothenate, and xanthine. In addition, four metabolic pathways are overlapping in group B vs. A and C vs. A; they are beta-alanine metabolism, biosynthesis of amino acids, linoleic acid metabolism, and aminoacyl-tRNA biosynthesis. These differential metabolites were involved in oxidative stress, immune function, energy metabolism, sugar metabolism, lipid metabolism, molecule transport, and weakened feed utilization, growth performance, nutrient metabolism, and animal growth. Furthermore, we found that the number of interfered amino acids and microplastics showed a dose-effect. In summary, great attention should be paid to the potential impact of microplastics on aquatic organisms.

Integrated analysis of tRNA-derived small RNAs in proliferative human aortic smooth muscle cells.

BACKGROUND: Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to vascular remodeling diseases. Recently, it has been discovered that tRNA-derived small RNAs (tsRNAs), a new type of noncoding RNAs, are related to the proliferation and migration of VSMCs. tsRNAs regulate target gene expression through miRNA-like functions. This study aims to explore the potential of tsRNAs in human aortic smooth muscle cell (HASMC) proliferation. METHODS: High-throughput sequencing was performed to analyze the tsRNA expression profile of proliferative and quiescent HASMCs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the sequence results and subcellular distribution of AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076. Based on the microRNA-like functions of tsRNAs, we predicted target promoters and mRNAs and constructed tsRNA-promoter and tsRNA-mRNA interaction networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to reveal the function of target genes. EdU incorporation assay, Western blot, and dual-luciferase reporter gene assay were utilized to detect the effects of tsRNAs on HASMC proliferation. RESULTS: Compared with quiescent HASMCs, there were 1838 differentially expressed tsRNAs in proliferative HASMCs, including 887 with increased expression (fold change > 2, p < 0.05) and 951 with decreased expression (fold change < ½, p < 0.05). AS-tDR-001370, AS-tDR-000067, AS-tDR-009512, and AS-tDR-000076 were increased in proliferative HASMCs and were mainly located in the nucleus. Bioinformatics analysis suggested that the four tsRNAs involved a variety of GO terms and pathways related to VSMC proliferation. AS-tDR-000067 promoted HASMC proliferation by suppressing p53 transcription in a promoter-targeted manner. AS-tDR-000076 accelerated HASMC proliferation by attenuating mitofusin 2 (MFN2) levels in a 3'-untranslated region (UTR)-targeted manner. CONCLUSIONS: During HASMC proliferation, the expression levels of many tsRNAs are altered. AS-tDR-000067 and AS-tDR-000076 act as new factors promoting VSMC proliferation.

A genetically engineered microRNA-34a prodrug demonstrates anti-tumor activity in a canine model of osteosarcoma.

Osteosarcoma (OSA) represents the most common primary bone tumor in humans and pet dogs. Little progress has been made with regard to viable treatment options in the past three decades and patients presenting with metastatic disease continue to have a poor prognosis. Recent mouse studies have suggested that microRNA-34a (miR-34a) may have anti-tumor activities in human OSA models. Due to the conservation of microRNA across species, we hypothesized that a bioengineered miR-34a prodrug (tRNA/miR-34a) would have similar effects in canine OSA, providing a valuable preclinical model for development of this therapeutic modality. Using a panel of canine OSA cell lines, we found that tRNA/miR-34a reduced viability, clonogenic growth, and migration and invasion while increasing tumor cell apoptosis. Furthermore, canine OSA cells successfully process the tRNA/miR-34a into mature miR-34a which reduces expression of target proteins such as platelet derived growth factor receptor alpha (PDGFRα), Notch1 and vascular endothelial growth factor (VEGF). Additionally, our subcutaneous OSA xenograft model demonstrated in vivo tumor growth delay, increased necrosis and apoptosis by tRNA/miR-34a, and decreased cellular proliferation ability. Taken together, these data support that this novel microRNA-based therapy may possess clinical utility in a spontaneously-occurring large animal model of OSA, which can then serve to inform the clinical development of this therapy for human OSA patients.

In vivo modification of tRNA with an artificial nucleobase leads to full disease remission in an animal model of multiple sclerosis.

Queuine is a modified pyrrolopyrimidine nucleobase derived exclusively from bacteria. It post-transcriptionally replaces guanine 34 in transfer RNA isoacceptors for Asp, Asn, His and Tyr, in almost all eukaryotic organisms, through the activity of the ancient tRNA guanine transglycosylase (TGT) enzyme. tRNA hypomodification with queuine is a characteristic of rapidly-proliferating, non-differentiated cells. Autoimmune diseases, including multiple sclerosis, are characterised by the rapid expansion of T cells directed to self-antigens. Here, we demonstrate the potential medicinal relevance of targeting the modification of tRNA in the treatment of a chronic multiple sclerosis model­murine experimental autoimmune encephalomyelitis. Administration of a de novo designed eukaryotic TGT substrate (NPPDAG) led to an unprecedented complete reversal of clinical symptoms and a dramatic reduction of markers associated with immune hyperactivation and neuronal damage after five daily doses. TGT is essential for the therapeutic effect, since animals deficient in TGT activity were refractory to therapy. The data suggest that exploitation of the eukaryotic TGT enzyme is a promising approach for the treatment of multiple sclerosis.

Inhibition of HIV-1 Gag-membrane interactions by specific RNAs.

HIV-1 particle assembly, which occurs at the plasma membrane (PM) of cells, is driven by the viral polyprotein Gag. Gag recognizes phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific phospholipid, via the highly basic region (HBR) in its N-terminal matrix (MA) domain. The HBR is also known to bind to RNA. We have previously shown, using an in vitro liposome binding assay, that RNA inhibits Gag binding to membranes that lack PI(4,5)P2 If this RNA block is removed by RNase treatment, Gag can bind nonspecifically to other negatively charged membranes. In an effort to identify the RNA species that confer this inhibition of Gag membrane binding, we have tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P2 We found that some tRNA species and RNAs containing stem-loop 1 of the psi region in the 5' untranslated region of the HIV-1 genome impose inhibition of Gag binding to membranes lacking PI(4,5)P2 In contrast, a specific subset of tRNAs, as well as an RNA sequence previously selected in vitro for MA binding, failed to suppress Gag-membrane interactions. Furthermore, switching the identity of charged residues in the HBR did not diminish the susceptibility of Gag-liposome binding for each of the RNAs tested, while deletion of most of the NC domain abrogates the inhibition of membrane binding mediated by the RNAs that are inhibitory to WT Gag-liposome binding. These results support a model in which NC facilitates binding of RNA to MA and thereby promotes RNA-based inhibition of Gag-membrane binding.

G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments.

Angiogenin (ANG) is a stress-activated ribonuclease that promotes the survival of motor neurons. Ribonuclease inactivating point mutations are found in a subset of patients with ALS, a fatal neurodegenerative disease with no cure. We recently showed that ANG cleaves tRNA within anticodon loops to produce 5'- and 3'-fragments known as tRNA-derived, stress-induced RNAs (tiRNAs). Selected 5'-tiRNAs (e.g., tiRNA(Ala), tiRNA(Cys)) cooperate with the translational repressor Y-box binding protein 1 (YB-1) to displace the cap-binding complex eIF4F from capped mRNA, inhibit translation initiation, and induce the assembly of stress granules (SGs). Here, we show that translationally active tiRNAs assemble unique G-quadruplex (G4) structures that are required for translation inhibition. We show that tiRNA(Ala) binds the cold shock domain of YB-1 to activate these translational reprogramming events. We discovered that 5'-tiDNA(Ala) (the DNA equivalent of 5'-tiRNA(Ala)) is a stable tiRNA analog that displaces eIF4F from capped mRNA, inhibits translation initiation, and induces the assembly of SGs. The 5'-tiDNA(Ala) also assembles a G4 structure that allows it to enter motor neurons spontaneously and trigger a neuroprotective response in a YB-1-dependent manner. Remarkably, the ability of 5'-tiRNA(Ala) to induce SG assembly is inhibited by G4 structures formed by pathological GGGGCC repeats found in C9ORF72, the most common genetic cause of ALS, suggesting that functional interactions between G4 RNAs may contribute to neurodegenerative disease.

Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

BACKGROUND: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. RESULTS: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. CONCLUSION: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

The 2'-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition.

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2'-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus-infected PBMCs.

A vast collection of microbial genes that are toxic to bacteria.

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract.

In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

A fungal tRNA of Aspergillus niger induces IFN-beta synthesis in HEp-2 cells.

In this work, we evaluated the capacity of a fungal transfer RNA (F-tRNA) from Aspergillus niger to protect HEp-2 cells against a viral infection, and as an inducer of IFN-beta synthesis. HEp-2 cells previously incubated with F-tRNA, polyI:polyC, or IFN-alpha, at different concentrations for 24 h were infected with 200 pfu of adenovirus type 6 (AdV-6); after 5 days, we determined cellular viability, cytopathic effect of the virus, optimal concentration necessary to inhibit the cytopathic effect, and IFN-beta expression by RT-PCR. Results showed that HEp-2 cells treated with F-tRNA were less susceptible to the cytopathic effect of AdV-6 infection than those incubated with polyI:polyC (p < 0.05). On the other hand, F-tRNA- treated HEp-2 cells expressed IFN-beta mRNA, whereas monolayers incubated with polyI:polyC or IFN-alpha did not. Our results suggest that F-tRNA protected HEp-2 cells against AdV-6 infection, due to its capacity to induce IFN-beta synthesis.

Reverse transcriptase of Moloney murine leukemia virus binds to eukaryotic release factor 1 to modulate suppression of translational termination.

The pol (for polymerase) gene of the murine leukemia viruses (MuLVs) is expressed in the form of a large Gag-Pol precursor protein by the suppression of translational termination, or enhanced readthrough, of a UAG stop codon at the end of gag. A search for cellular proteins that interact with the reverse transcriptase of Moloney MuLV resulted in the identification of eRF1, the eukaryotic translation release factor 1. The proteins bound strongly in vitro, and the overexpression of eRF1 resulted in the RT-dependent incorporation of the protein into assembling virion particles. The overexpression of RT in trans enhanced the translational readthrough of a reporter construct containing the Gag-Pol boundary region. Noninteracting mutants of RT failed to synthesize adequate levels of Gag-Pol and could not replicate. These results suggest that RT enhances suppression of termination and that the interaction of RT with eRF1 is required for an appropriate level of translational readthrough.

A spectrophotometric method to quantify linear DNA.

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA.

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.

A Ran-independent pathway for export of spliced mRNA.

All major nuclear export pathways so far examined follow a general paradigm. Specifically, a complex is formed in the nucleus, containing the export cargo, a member of the importin-beta family of transporters and RanGTP. This complex is translocated across the nuclear pore to the cytoplasm, where hydrolysis of the GTP on Ran is stimulated by the GTPase-activating protein RanGAP. The activity of RanGAP is increased by RanBP1, which also promotes disassembly of RanGTP-cargo-transporter complexes. Here we investigate the role of RanGTP in the export of mRNAs generated by splicing. We show that nuclear injection of a Ran mutant (RanT24N) or the normally cytoplasmic RanGAP potently inhibits the export of both tRNA and U1 snRNA, but not of spliced mRNAs. Moreover, nuclear injection of RanGAP together with RanBP1 blocks tRNA export but does not affect mRNA export. These and other data indicate that export of spliced mRNA is the first major cellular transport pathway that is independent of the export co-factor Ran.