Zeitschriftenartikel zum Thema „Thiouridylase“

All kingdoms of life have more than 150 different forms of RNA alterations, with tRNA accounting for around 80% of them. These chemical alterations include, among others, methylation, sulfuration, hydroxylation, and acetylation. These changes are necessary for the proper codon recognition and stability of tRNA. In Escherichia coli, sulfur modification at the wobble uridine (34) of lysine, glutamic acid, and glutamine is essential for codon and anticodon binding and prevents frameshifting during translation. Two important proteins that are involved in this thiolation modification are the L-cysteine desulfurase IscS, the initial sulfur donor, and tRNA-specific 2-thiouridylase MnmA, which adenylates and finally transfers the sulfur from IscS to the tRNA. tRNA-specific 2-thiouridylases are iron–sulfur clusters (Fe-S), either dependent or independent depending on the organism. Here, we dissect the controversy of whether the E. coli MnmA protein is an Fe-S cluster-dependent or independent protein. We show that when Fe-S clusters are bound to MnmA, tRNA thiolation is inhibited, making MnmA an Fe-S cluster-independent protein. We further show that 2-thiouridylase only binds to tRNA from its own organism.

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

Мутации в гене TRMU, кодирующем одну из митохондриальных тРНК метилтрансфераз, были обнаружены при инфантильной гепатопатии, связанной с дефектом митохондриальной трансляции (OMIM#613070). Это заболевание является редким заболеванием с угрожающим жизни началом и во многих случаях с последующей спонтанной ремиссией. Своевременная диагностика и лечение таких больных имеют важное значение в клинической практике. В статье приводится описание пациента с печеночной недостаточностью, обусловленной мутациями в гене TRMU, и сравнение клинической картины с литературными данными. Mutations in the TRMU gene encoding the mitochondrial tRNA-specific 2-thiouridylase were found in infantile hepatopathy related to mitochondrial translation defect (OMIM# 613070). This condition is rare mitochondrial disorder with a life-threatening onset and with spontaneous remission, therefore a prompt diagnosis and treatment of these patients has importance in clinical practice. Here we describe a patient, with liver failure due to mutations in TRMU gene and compare with patients from literature.

Abstract Evasion of apoptosis is a hallmark of cancer wherein overexpression and amplification of pro-survival BCL2-family genes like MCL1 is a common observation. MCL1 is frequently amplified in many hematological cancers like Multiple Myeloma (MM) that depend on it for survival. BH3 mimetic drugs, like the BCL2-specific inhibitor Venetoclax, have been successfully used in the clinic to treat certain cancers, and MCL1-selective inhibitors are currently in clinical development. While inhibition of MCL1 displays promising preclinical activity, many cancer models display acquired or intrinsic resistance to MCL1 inhibitors (MCL1i). As MCL1-targeted therapies progress clinically, understanding mechanisms that lead to resistance will be important to not only identify therapeutically-exploitable targets to combat resistance, but to also determine if these biomarkers could stratify patients most likely to respond to an MCL1i. Here, we used a genome-wide CRISPR knock-out screen to identify mechanisms of resistance to MCL1i AZD5991 in two MM cell lines, KMS11 and KMS34. We used a sgRNA library consisting of about 118,000 sgRNAs (~6 sgRNAs/gene), and treated the cells with DMSO or 1uM AZD5991 for 16 days (5 doublings). We identified 316 genes in KMS11 and 184 genes in KMS34 with >4-fold enrichment of sgRNAs; and 221 genes with >2-fold enrichment of sgRNAs in both cell lines. The sgRNAs targeting BAK and BAX were the most enriched overlapping hits. Using GSEA analysis of the 221 common genes with enriched sgRNAs, we discovered that the tRNA wobble uridine modification as the most enriched pathway. The tRNA U34 mcm5s2 modification is catalyzed by the elongator complex ELP1-6 and cytosolic thiouridylase CTU1/2. Each subunit of the elongator complex is essential for its function and loss of any subunit results in destabilization of the complex. By knocking out ELP4 in five MM cell lines (KMS11, KMS34, KMS12-PE, MM.1S, and RPMI-8226), we first validated the destabilization of the complex by showing a robust decrease in the protein levels of ELP1 and ELP3 via western blot. As the elongator complex has additional functions, we also knocked-out tRNA U34 modification pathway specific CTU1 in KMS11, KMS34, and KMS12-PE cells. We showed that genetic knock-out of ELP4 and CTU1 results in increased resistance to MCL1i in all cell lines tested. We observed the highest increase in MCL1i resistance upon ELP4-KO in KMS11 and RPMI-8226 cell lines. To understand the mechanism behind elongator complex mediated regulation of MCL1 dependency, we performed RNAseq and global proteomics in KMS11 cells (Parental, non-targeting control [NTC], ELP4-KO and CTU1-KO) and RPMI-8226 cells (Parental, NTC, and ELP4-KO). We show that the elongator complex is a regulator of IRE1-XBP1 axis of the ER stress response pathway; and knockout of IRE1 also results in MCL1i-resistance in KMS11 and RPMI8226 cell lines. Mechanistically, we show that loss of elongator complex-mediated downregulation of IRE1-XBP1 axis leads to stabilization of MCL1 and upregulation of BCL-XL and NOXA expression. We further show that upon treatment with MCL1i, KMS11-ELP4-KO cells have less disruption of MCL1:Bim complex and an increase in BCL-XL:Bim complex as compared with KMS11-NTC cells. The net increase in pro-survival MCL1 and BCL-XL proteins in ELP4-KO cells resulting in lower levels of unsequestered BIM upon AZD5991 treatment suggests a reduction in apoptotic priming. The mechanistic link between the elongator complex and ER stress response pathway led us to test ER stress inducing drugs in these cell lines. We observed that ELP4-KO results in increased resistance to proteasome inhibitor Bortezomib and other ER stress inducers like Tunicamycin, Thapsigargin, and BrefeldinA as a monotherapy or in combination with AZD5991. These data are consistent with our hypothesis that ELP4-KO cells have reduced apoptotic priming and are thus multi-drug resistant. As bortezomib is used in the clinic to treat MM patients, we asked if an elongator gene signature could be used to predict response to current therapies. We show that the elongator complex components could be used as a gene signature to stratify overall survival in MM patients (MMRF CoMMpass dataset). Moreover, ER stress response gene signature has been shown to be repressed in drug-resistant MM. Taken together, an integrated elongator and IRE-XBP1 gene signature could be a strong predictor of therapy response in MM . Disclosures Aryal: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Sundarrajan: AstraZeneca: Ended employment in the past 24 months. Boiko: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jenkins: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Liu: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Ahdesmaki: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Bornot: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Jarnuczak: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Miele: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. McDermott: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Fawell: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Drew: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Boise: AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

Toxoplasmosis caused by the protozoan Toxoplasma gondii is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs contain a super-modified 2-thiourea (s2U) derivatives at the position 34, which is essential for all living organisms by maintaining the structural stability and aminoacylation of tRNA, and the precision and efficiency of codon recognition during protein translation. However, the enzyme(s) involved in this modification in T. gondii remains elusive. In this report, three putative tRNA-specific 2-thiolation enzymes were identified, of which two were involved in the s2U34 modification of tRNALys, tRNAGlu, and tRNAGln. One was named TgMnmA, an apicoplast-located tRNA-specific 2-thiolation enzyme in T. gondii. Knockout of TgMnmA showed that this enzyme is important for the lytic cycle of tachyzoites. Loss of TgMnmA also led to abnormities in apicoplast biogenesis and severely disturbed apicoplast genomic transcription. Notably, mice survived from the infection with 10 TgMnmA-KO RH tachyzoites. These findings provide new insights into s2U34 tRNA modification in Apicomplexa, and suggest TgMnmA, the first apicoplast tRNA thiouridylase identified in all apicomplexans, as a potential drug target.

AbstractPlasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Abstract MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

AbstractCytosolic thiouridylase 2 (CTU2) is an enzyme modifying transfer RNAs post-transcriptionally, which has been implicated in breast cancer and melanoma development. And we found CTU2 participated in hepatocellular carcinoma (HCC) progression here. HepG2 cells as well as xenograft nude mice model were employed to investigate the role of CTU2 in HCC development in vitro and in vivo respectively. Further, we defined CTU2 as a Liver X receptor (LXR) targeted gene, with a typical LXR element in the CTU2 promoter. CTU2 expression was activated by LXR agonist and depressed by LXR knockout. Interestingly, we also found CTU2 took part in lipogenesis by directly enhancing the synthesis of lipogenic proteins, which provided a novel mechanism for LXR regulating lipid synthesis. Meanwhile, lipogenesis was active during cell proliferation, particularly in tumor cells. Reduction of CTU2 expression was related to reduced tumor burden and synergized anti-tumor effect of LXR ligands by inducing tumor cell apoptosis and inhibiting cell proliferation. Taken together, our study identified CTU2 as an LXR target gene. Inhibition of CTU2 expression could enhance the anti-tumor effect of LXR ligand in HCC, identifying CTU2 as a promising target for HCC treatment and providing a novel strategy for the application of LXR agonists in anti-tumor effect. Graphical Abstract 1.) CTU2 enhances proliferation of hepatoma carcinoma cells. 2.) CTU2 is the target gene of LXR, and LXR can transcriptionally activate CTU2 expression. 3.) CTU2 can promote protein synthesis of lipogenic genes. 4.) Inhibiting CTU2 expression can synergistically enhance the inhibitory effects of LXR ligands on HCC growth.

Post‐transcriptional modifications of tRNA are found in all organisms and are critical for their structure and function during translation. 2‐thiouridine (s2U) modification at the wobble position of tRNAGlu, Gln, Lys is required for accurate and efficient protein synthesis and its absence impacts cellular viability. In addition to its role in translation, in yeast, the s2U modification is used as a sensor to gauge nutrient availability and regulate translational capacity and amino acid homeostasis, in which s2U abundance reflects the availability of sulfur‐containing amino acids, cysteine and methionine. In previous work, we showed that B. subtilis s2U biosynthesis involves only two proteins: the cysteine desulfurase YrvO and the thiouridylase MnmA. Genes coding for both proteins are located in an operon directly downstream of the master regulator of cysteine metabolism, cymR, suggesting a link between s2U formation and sulfur amino acid metabolism in this bacterium.In this study we investigated s2U's role in metabolic homeostasis and translational regulation as a sensor of sulfur availability in bacteria. B. subtilis wild‐type was cultured in minimal media containing varying concentrations of an individual sulfur source: sulfate, cysteine, or methionine. Using our established LC‐MS method, analysis of tRNA‐thionucleosides showed that the abundance of s2U correlates to the concentration, but not the source, of sulfur available in the growth media. This relationship appears to be specific to s2U, as the levels of an additional thionucleoside, s4U, remained constant across all conditions; supporting the notion that only s2U is responsive to sulfur availability. Western blot analysis of YrvO and MnmA in these cultures showed patterns consistent with those of CymR expression. That is, higher expression of YrvO and MnmA was observed in cultures using sulfate, cysteine or cystine as the sole sulfur source when compared to thiosulfate, methionine or homocystine. Surprisingly, expression levels of YrvO and MnmA did not correlate with the concentration of sulfur source, or accumulation of s2U thionucleoside, suggesting that the activity of these enzymes is likely controlled by the concentration of cysteine, which affects the catalytic turnover rate of YrvO, the first enzyme in the s2U pathway. In this model, the availability of sulfur controls the flux of sulfur transfer which impacts the overall levels of the signaling metabolite s2U tRNA. Current work is aimed towards evaluating whether s2U levels dictate expression of proteins involved in translation and growth specific processes. Furthermore, these analyses are being expanded to other bacterial species to potentially establish this regulatory role across a diverse group of bacteria.Support or Funding InformationThis work has been funded through the Wake Forest University Center for Molecular Communication and Signaling graduate fellowship.