Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

Uremic Solute-Aryl Hydrocarbon Receptor-Tissue Factor Axis Associates with Thrombosis after Vascular Injury in Humans.

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37.

Post-transcriptional modifications of anticodon loop (ACL) nucleotides impact tRNA structure, affinity for the ribosome, and decoding activity, and these activities can be fine-tuned by interactions between nucleobases on either side of the anticodon. A recently discovered ACL modification circuit involving positions 32, 34, and 37 is disrupted by a human disease-associated mutation to the gene encoding a tRNA modification enzyme. We used tRNA-HydroSeq (-HySeq) to examine (3)methyl-cytidine-32 (m(3)C32), which is found in yeast only in the ACLs of tRNAs(Ser) and tRNAs(Thr) In contrast to that reported for Saccharomyces cerevisiae in which all m(3)C32 depends on a single gene, TRM140, the m(3)C32 of tRNAs(Ser) and tRNAs(Thr) of the fission yeast S. pombe, are each dependent on one of two related genes, trm140(+) and trm141(+), homologs of which are found in higher eukaryotes. Interestingly, mammals and other vertebrates contain a third homolog and also contain m(3)C at new sites, positions 32 on tRNAs(Arg) and C47:3 in the variable arm of tRNAs(Ser) More significantly, by examining S. pombe mutants deficient for other modifications, we found that m(3)C32 on the three tRNAs(Ser) that contain anticodon base A36, requires N(6)-isopentenyl modification of A37 (i(6)A37). This new C32-A37 ACL circuitry indicates that i(6)A37 is a pre- or corequisite for m(3)C32 on these tRNAs. Examination of the tRNA database suggests that such circuitry may be more expansive than observed here. The results emphasize two contemporary themes, that tRNA modifications are interconnected, and that some specific modifications on tRNAs of the same anticodon identity are species-specific.

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA.

tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N(6)-A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. Schizosaccharomyces pombe IPTase modifies three different cy-tRNAs(Ser) as well as cy-tRNA(Tyr), cy-tRNA(Trp), and mt-tRNA(Trp). We show that lower ATP levels in tit1-Δ relative to tit1(+) cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA(Tyr) alone substantially does so. Bioinformatics indicate that cy-tRNA(Tyr) is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodification-associated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA(Tyr), and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.

RNA polymerase III mutants in TFIIFα-like C37 that cause terminator readthrough with no decrease in transcription output.

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFß/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3'-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3'-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3'-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3'-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and (14)C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription.

Transcription termination by the eukaryotic RNA polymerase III.

RNA polymerase (pol) III transcribes a multitude of tRNA and 5S rRNA genes as well as other small RNA genes distributed through the genome. By being sequence-specific, precise and efficient, transcription termination by pol III not only defines the 3' end of the nascent RNA which directs subsequent association with the stabilizing La protein, it also prevents transcription into downstream DNA and promotes efficient recycling. Each of the RNA polymerases appears to have evolved unique mechanisms to initiate the process of termination in response to different types of termination signals. However, in eukaryotes much less is known about the final stage of termination, destabilization of the elongation complex with release of the RNA and DNA from the polymerase active center. By comparison to pols I and II, pol III exhibits the most direct coupling of the initial and final stages of termination, both of which occur at a short oligo(dT) tract on the non-template strand (dA on the template) of the DNA. While pol III termination is autonomous involving the core subunits C2 and probably C1, it also involves subunits C11, C37 and C53, which act on the pol III catalytic center and exhibit homology to the pol II elongation factor TFIIS and TFIIFα/ß respectively. Here we compile knowledge of pol III termination and associate mutations that affect this process with structural elements of the polymerase that illustrate the importance of C53/37 both at its docking site on the pol III lobe and in the active center. The models suggest that some of these features may apply to the other eukaryotic pols. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III.

RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3' oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3'-5' cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA-DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3' cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination.

Diagnostic value of pleural cholesterol in differentiating exudative and transudative pleural effusion.

Background: Pleural effusions are most commonly classified as transudative or exudative based on Light's criteria which has shown misclassification in 10%-20% of cases. Studies have demonstrated lesser misclassification with pleural fluid cholesterol criteria. Thus, this study aimed to find the diagnostic properties of pleural fluid cholesterol in differentiating the type of effusion. Materials and methods: This cross-sectional study involving 72 patients was undertaken in a tertiary center in Nepal for a duration of 2 years. On the basis of Light's, Heffner's, etiological, and pleural fluid cholesterol criteria, pleural effusion was classified as exudative or transudative. The findings were then evaluated to determine the diagnostic value of each approach in identifying the effusion type and comparing them on the basis of sensitivity, specificity, positive predictive value and negative predictive value. Result: Pleural fluid cholesterol detected effusion as exudative with sensitivity of 91.94% and specificity of 80.00% against Light's criteria; with a sensitivity of 98.28% and specificity of 85.71% against the etiological diagnosis. Additionally, against the etiological diagnosis, sensitivity of both Light's and Heffner's criteria was 100%; however, specificity was 71.43% and 64.29% respectively, which is far less than that of pleural fluid cholesterol (85.71%). Furthermore, pleural fluid cholesterol was also found to have better results than protein ratio, LDH ratio and pleural fluid protein ratio in determining the type of effusion. Conclusion: When considering the avoidance of confusing outcomes in equivocal instances and cost effectiveness in developing nations, pleural fluid cholesterol can be one of the most useful alternative diagnostic methods for differentiating between exudative or transudative effusions.

A new role for cisplatin: probing ribosomal RNA structure.

In this study, cisplatin, the well-known anticancer drug, was used to probe accessible purine residues in E. coli 16S ribosomal RNA (rRNA), free or in the context of 30S subunits or 70S ribosomes, both in vitro and in vivo.

A mass spectrometric method for quantification of tryptophan-derived uremic solutes in human serum.

In addition to various physiologic roles, emerging evidence strongly points to pathogenic roles of tryptophan and of its metabolites, especially in diseases such as renal failure. Accurate estimation of levels of these metabolites in blood is important to mechanistically probe their contribution to disease pathogenesis, while clinically, such a panel can be used to risk stratify patients for a clinical phenotype. Herein, we describe a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based method to determine the level of tryptophan and its metabolites (kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate and indoxyl acetate). Human sera samples were processed through a C18 column followed by application of a binary gradient and quantitation by MS/MS. The linearity, lower limit of detection, inter- and intraassay variabilities and recovery were determined, yielding a precise, reproducible method for all the metabolites. Unlike previous studies, we further validated these methods in a well-characterized set of human sera from end stage renal disease patients compared to age-, gender- and ethnic-background matched human controls. Overall, we report an optimized LC/MS-based estimation of a comprehensive panel of tryptophan-derived metabolites with quality features within FDA standards, underscoring their readiness for translational use.

Targeting STUB1-tissue factor axis normalizes hyperthrombotic uremic phenotype without increasing bleeding risk.

Chronic kidney disease (CKD/uremia) remains vexing because it increases the risk of atherothrombosis and is also associated with bleeding complications on standard antithrombotic/antiplatelet therapies. Although the associations of indolic uremic solutes and vascular wall proteins [such as tissue factor (TF) and aryl hydrocarbon receptor (AHR)] are being defined, the specific mechanisms that drive the thrombotic and bleeding risks are not fully understood. We now present an indolic solute-specific animal model, which focuses on solute-protein interactions and shows that indolic solutes mediate the hyperthrombotic phenotype across all CKD stages in an AHR- and TF-dependent manner. We further demonstrate that AHR regulates TF through STIP1 homology and U-box-containing protein 1 (STUB1). As a ubiquitin ligase, STUB1 dynamically interacts with and degrades TF through ubiquitination in the uremic milieu. TF regulation by STUB1 is supported in humans by an inverse relationship of STUB1 and TF expression and reduced STUB1-TF interaction in uremic vessels. Genetic or pharmacological manipulation of STUB1 in vascular smooth muscle cells inhibited thrombosis in flow loops. STUB1 perturbations reverted the uremic hyperthrombotic phenotype without prolonging the bleeding time, in contrast to heparin, the standard-of-care antithrombotic in CKD patients. Our work refines the thrombosis axis (STUB1 is a mediator of indolic solute-AHR-TF axis) and expands the understanding of the interconnected relationships driving the fragile thrombotic state in CKD. It also establishes a means of minimizing the uremic hyperthrombotic phenotype without altering the hemostatic balance, a long-sought-after combination in CKD patients.

A methods review on use of nonsense suppression to study 3' end formation and other aspects of tRNA biogenesis.

Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to Schizosaccharomyces pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5' and 3' processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3' oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3' oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a 'technical approaches' section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses.

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation.

In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.

Amino acid-linked platinum(II) analogues have altered specificity for RNA compared to cisplatin.

Cisplatin can be modified with various ligands to alter the size and charge distribution of the complex. Several amino acid-linked platinum(II) complexes were synthesized, and a reactivity study with 16S ribosomal RNA was carried out. The amino acid-linked analogues show altered specificity compared to the parental compound cisplatin.

CT evaluation of pelvic and hip fractures.

INTRODUCTION: To analyze the spectrum of pelvic and hip joint injuries by computed tomography in patients attending to emergency. METHODS: CT evaluation with multiplanar and 3-D reformations of 62 patients done with pelvic and hip fractures during 18 months. The mode of injury and treatment were recorded. RESULTS: Forty four (70.9%) of the injuries were sustained in high speed vehicle accidents (automobile, motorcycle, autopedestrian), 14 patients (22.5%) had sustained fall injury and 4 patients (6.4%) sustained injury following slip on the ground. 31 patients underwent surgery for acetabular fractures. Associated injuries included spinal fractures (5), abdominal visceral injury (3), extremity fractures (13), chest injury (5), head injury (1), urethral injury (4), clavicle fracture (1). Forty patients (64.5%) had acetabular fractures, 25 (40.3%) patients had iliac bone fractures, 47 (75.8%) had pubic bone fracture. Sacral fracture was found in 17 (27.4%) patients. Associated femoral fracture was found in 4, hip joint dislocation was seen in 5 (2- central, 3-posterior). Sacroiliac joint diastasis was found in 13(20.9%) patients, pubic diastasis in 7 (11.2%), fracture ischium in 7(11.%), protrusio acetabuli in 5 (8%). CONCLUSIONS: Spiral CT with multiplanar and 3-D reformations has clinical values for precise diagnosis and treatment plan for the pelvic and hip fractures.