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RNA terminal phosphorylase B (RTCB) has been shown to play a significant role in multiple physiological processes. However, the specific role of RTCB in the mouse colon remains unclear. In this study, we employ a conditional knockout mouse model to investigate the effects of RTCB depletion on the colon and the potential molecular mechanisms. We assess the efficiency and phenotype of Rtcb knockout using PCR, western blot analysis, histological staining, and immunohistochemistry. Compared with the control mice, the Rtcb-knockout mice exhibit compromised colonic barrier integrity and prominent inflammatory cell infiltration. In the colonic tissues of Rtcb-knockout mice, the protein levels of TNF-α, IL-8, and p-p65 are increased, whereas the levels of IKKß and IκBα are decreased. Moreover, the level of GSK3ß is increased, whereas the levels of Wnt3a, ß-catenin, and LGR5 are decreased. Collectively, our findings unveil a close association between RTCB and colonic tissue homeostasis and demonstrate that RTCB deficiency can lead to dysregulation of both the NF-κB and Wnt/ß-catenin signaling pathways in colonic cells.
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Colitis , FN-kappa B , Animales , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Colitis/genética , Ratones Noqueados , FN-kappa B/metabolismo , Vía de Señalización WntRESUMEN
To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.
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ARN Ligasa (ATP) , Factores de Transcripción , Humanos , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/química , ARN Ligasa (ATP)/metabolismo , Factores de Transcripción/metabolismo , ARN/metabolismo , Respuesta de Proteína Desplegada , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Empalme del ARN/genéticaRESUMEN
Under stress conditions, transfer RNAs (tRNAs) are cleaved by stress-responsive RNases such as angiogenin, generating tRNA-derived RNAs called tiRNAs. As tiRNAs contribute to cytoprotection through inhibition of translation and prevention of apoptosis, the regulation of tiRNA production is critical for cellular stress response. Here, we show that RTCB ligase complex (RTCB-LC), an RNA ligase complex involved in endoplasmic reticulum (ER) stress response and precursor tRNA splicing, negatively regulates stress-induced tiRNA production. Knockdown of RTCB significantly increased stress-induced tiRNA production, suggesting that RTCB-LC negatively regulates tiRNA production. Gel-purified tiRNAs were repaired to full-length tRNAs by RtcB in vitro, suggesting that RTCB-LC can generate full length tRNAs from tiRNAs. As RTCB-LC is inhibited under oxidative stress, we further investigated whether tiRNA production is promoted through the inhibition of RTCB-LC under oxidative stress. Although hydrogen peroxide (H2O2) itself did not induce tiRNA production, it rapidly boosted tiRNA production under the condition where stress-responsive RNases are activated. We propose a model of stress-induced tiRNA production consisting of two factors, a trigger and booster. This RTCB-LC-mediated boosting mechanism may contribute to the effective stress response in the cell.
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Peróxido de Hidrógeno , ARN de Transferencia , Peróxido de Hidrógeno/farmacología , ARN de Transferencia/metabolismo , Estrés Oxidativo , Empalme del ARN , Ligasas/genéticaRESUMEN
Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].
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Perfilación de la Expresión Génica , ARN Catalítico/genética , RNA-Seq/métodos , Arabidopsis/genética , Biología Computacional , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Genómica/métodos , ARN Catalítico/químicaRESUMEN
The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.
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The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.
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Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , ARN Ligasa (ATP)/metabolismo , ARN de Transferencia/metabolismo , Animales , Antioxidantes/fisiología , Dominio Catalítico , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/fisiología , ARN Ligasa (ATP)/química , ARN Ligasa (ATP)/genética , Empalme del ARN/genética , Empalme del ARN/fisiología , Respuesta de Proteína Desplegada/fisiología , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
HSPC117/RtcB, 3'-phosphate tRNA ligase, is a critical enzyme involved in tRNA splicing and maturation. HSPC117/RtcB is also involved in mRNA splicing of some protein-coding genes including XBP-1. Entamoeba histolytica, a protozoan parasite responsible for human amebiasis, possesses two RtcB proteins (EhRtcB1 and 2), but their biological functions remain unknown. Both RtcBs show kinship with mammalian/archaeal type, and all amino acid residues present in the active sites are highly conserved, as suggested by protein alignment and phylogenetic analyses. EhRtcB1 was demonstrated to be localized to the nucleus, while EhRtcB2 was in the cytosol. EhRtcB1, but not EhRtcB2, was required for optimal growth of E. histolytica trophozoites. Both EhRtcB1 (in cooperation with EhArchease) and EhRtcB2 showed RNA ligation activity in vitro. The predominant role of EhRtcB1 in tRNAIle(UAU) processing in vivo was demonstrated in EhRtcB1- and 2-gene silenced strains. Taken together, we have demonstrated the conservation of tRNA splicing and functional diversification of RtcBs in this amoebozoan lineage.
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Entamoeba histolytica , ARN Ligasa (ATP) , Animales , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Humanos , Fosfatos , Filogenia , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , Empalme del ARNRESUMEN
Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5'-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2'-, 3'-cyclic phosphate groups, as part of a new CLIP method that does not require 5'-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3'-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.
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MicroARNs/química , MicroARNs/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Ligasas/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.
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Operón/inmunología , ARN de Transferencia/metabolismo , ARN/metabolismo , HumanosRESUMEN
Genistein, a soy-derived phytoestrogen, has been shown to exhibit anti-neoplastic activities in various cancers. Nevertheless, its effects on the elimination of tumor-initiating cells of head and neck cancer (HNC-TICs) remain unclear. Here, we investigated the inhibitory effect of genistein on HNC-TICs and potential mechanisms. Our results demonstrated that genistein lowered the proliferation of HNC-TICs by examining the percentage of ALDH1+ or CD44+ cells. Aside from the downregulation of epithelial-mesenchymal transition (EMT) in HNC-TICs, genistein restricted their tumor propagating capacities in a dose-dependent fashion. Moreover, genistein potentiated cell death caused by three commonly used chemotherapeutic agents (doxorubicin, cisplatin, and 5-FU). Our findings proved that genistein induced ROS production through upregulation of miR-34a, leading to apoptosis in HNC-TICs. The genistein-elicited miR-34a reduced self-renewal, migration, invasion capacities and ALDH1 activity, which may be partly owing to the repression of EMT. Furthermore, we showed that RTCB was a novel target that was negatively regulated by miR-34a and involved in the tumor repressive effect of genistein. Besides, the in vivo study validated that genistein retarded tumor growth through the elevation of miR-34a and suppression of RTCB. These results suggested that genistein-induced miR-34a contributed to the ROS-associated apoptosis and diminished stemness properties via repression of RTCB in HNC-TICs.
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Anticarcinógenos/farmacología , Genisteína/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , MicroARNs/metabolismo , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Familia de Aldehído Deshidrogenasa 1 , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Humanos , Receptores de Hialuranos , Células Madre Neoplásicas/efectos de los fármacosRESUMEN
The human archease, hereafter named HArch, is identified as a key cofactor of the tRNA-splicing ligase complex, and a potential therapeutic target for treating nervous system injuries. However, little is known about the structural basis of HArch in tRNA maturation, mRNA splicing, and RNA repair. Here we report the crystal structures of HArch and its two mutants D51A and D178A with resolutions ranging from 1.96 Å to 3.4 Å. HArch is composed of an extended N-terminal protrusion domain (NTD) and one compacted C-terminal domain (CTD). Unlike previously reported homologous proteins, the NTD of the first subunit interacts with the CTD of the second one, and this interaction might be important for maintaining protein stability. Moreover, HArch interacts and colocalizes with RNA ligase RTCB in cells. Our current study reveals the atomic structure of HArch and may help us understand its function in mRNA splicing.
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ARN Ligasa (ATP)/química , Proteínas de Unión al ARN/química , Cristalografía por Rayos X , Humanos , ARN Ligasa (ATP)/metabolismo , Proteínas de Unión al ARN/metabolismo , TransfecciónRESUMEN
PURPOSE: To investigate the association between the lncRNA NEAT1 and breast cancer, and to determine the influence of NEAT1 on regulation of other signaling molecules in breast cancer. METHODS: In the present study, we measured levels of the lncRNA NEAT1 in 106 breast cancer patients and in a human breast cancer cell line by qRT-PCR. The correlation between NEAT1 expression and patients' clinical characteristics was analyzed with in-house and TCGA data. We used cellular functioning assays and cell immunofluorescence assay to evaluate the role of NEAT1 and its target molecules in proliferation, invasion and migration in breast cancer. We used Western blotting to explore possible targets of NEAT1 and a subcellular fractionation assay to locate NEAT1 expression. RESULTS: NEAT1 was overexpressed in breast cancer tissue and also closely related to advanced clinical stages and positive lymph node metastases. NEAT1 levels were also tightly correlated to prognosis for breast cancer patients in survival analyses. Cellular function assays revealed that downregulation of NEAT1 could inhibit breast cancer cell viability, invasion and migration. Western blotting revealed down-regulation of CBX7 and up-regulation of RTCB following NEAT1 inhibition. Based on the cytoplasmic and nuclear expression of NEAT1, we investigated the possible regulation of CBX7 and RTCB by NEAT1. Results showed that NEAT1 regulated the expression of CBX7 and RTCB, possibly by binding of NEAT1 to DNA in the nucleus, which facilitates cell proliferation, invasion and migration. CONCLUSION: The current results suggest that the lncRNA NEAT1 is upregulated in breast cancer and facilitates tumor cell viability, invasion and migration via CBX7 and RTCB.
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RtcB, a highly conserved RNA ligase, is found in all three domains of life, and demonstrated to be an essential tRNA splicing component in archaea and metazoans. However, the biological functions of RtcB in bacteria, where there is no splicing, remains to be clarified. We first performed bioinformatics analysis which revealed highly conserved structures and presumably conserved functions of RtcB in bacteria. However, its orthologs only occur in â¼ 0.5% of bacterial species across diverse phyla with significant signals of frequent horizontal transfer, highlighting its non-essential role in bacteria. Next, by constructing an rtcB-knockout strain, we find that the removal of antibiotic stress induces a significant impact on rtcB expression in wild-type strain, and furthermore the depletion of RtcB (ARtcB strain) delays the recovery process. Our transcriptomic analysis, comprising the 3'-end labeling of RNAs, highlights a significant increase in unmapped reads and cleaved rRNAs in the Δ RtcB strain, particularly during recovery. Our observations suggest that the conserved RNA ligase RtcB, repairs damaged rRNAs following stress, which potentially saves energy and accelerates recovery of its host. We propose that acquisition of RtcB by diverse bacterial taxa provides a competitive advantage under stressful conditions.
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Aminoacil-ARNt Sintetasas/genética , Antibacterianos/metabolismo , Proteínas de Escherichia coli/genética , ARN Ligasa (ATP)/genética , ARN Bacteriano/metabolismo , Estrés Fisiológico/genética , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Mutación/genética , ARN Ligasa (ATP)/metabolismo , Empalme del ARN/genética , ARN de Transferencia/metabolismo , Homología de SecuenciaRESUMEN
The presence of introns in both protein-coding and noncoding RNA transcripts is a fascinating phenomenon. It seems counterintuitive that an organism would devote precious time and energy to removing a nucleic acid sequence that will not be present in the final product. Nevertheless, introns (including self-splicing ones) are clearly important components of the basic cellular process of gene expression. Transfer RNA (tRNA) introns have been detected in all three kingdoms of life, and their precise removal is crucial for tRNA function. Of particular interest to this review are the tRNA intronic circular RNAs (tricRNAs) that form during metazoan tRNA splicing. In animal cells, these ultrastable introns form a novel class of noncoding RNA. Here, we summarize established knowledge and describe new findings in the field of tRNA splicing. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA in Disease and Development > RNA in Disease RNA Processing > tRNA Processing.
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ARN de Transferencia/genética , Animales , Humanos , Intrones , Conformación de Ácido Nucleico , Empalme del ARN/genética , ARN no Traducido/genéticaRESUMEN
Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind their encoding transcript to promote retrotransposition in cis The L1-encoded proteins also promote the retrotransposition of small-interspersed element RNAs, noncoding RNAs, and messenger RNAs in trans Some L1-mediated retrotransposition events consist of a copy of U6 RNA conjoined to a variably 5'-truncated L1, but how U6/L1 chimeras are formed requires elucidation. Here, we report the following: The RNA ligase RtcB can join U6 RNAs ending in a 2',3'-cyclic phosphate to L1 RNAs containing a 5'-OH in vitro; depletion of endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation efficiency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene formation; and a unique cohort of U6/L1 chimeric RNAs are present in multiple human cell lines. Thus, these data suggest that U6 small nuclear RNA (snRNA) and RtcB participate in the formation of chimeric RNAs and that retrotransposition of chimeric RNA contributes to interindividual genetic variation.
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Células Madre Embrionarias/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias/genética , Células-Madre Neurales/metabolismo , ARN Nuclear Pequeño/genética , ARN/genética , Retroelementos/genética , Células HeLa , Humanos , Seudogenes , ARN/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Nuclear Pequeño/químicaRESUMEN
OBJECTIVE: To identify the key residues of Thermus thermophilus (T. thermophilus) RTCB in RNA ligation and DNA activation. RESULTS: The biochemical activities of RTCB from T. thermophilus were purified, characterized, and compared. Structure and sequence alignment between T. thermophilus RTCB and Pyrococcus horikoshii (P. horikoshii) RTCB identified six conserved residues (D64, D95, N203, H204, E207, H399) that were essential for RNA ligation. Mutation analysis showed that the expression levels of mutants D95A, N203A, H204A, E207A and H399A were relatively low. Compared to wide-type RTCB, variant D64A protein had no RNA ligation and DNA activation activity. In addition, T. thermophilus RTCB showed acceptable catalytic activity of 3'-phosphate DNA activation at 37 °C. CONCLUSION: D64 was proved to be essential for RTCB-catalyzed RNA ligation and DNA activation (from 37 to 70 °C) in T. thermophilus.
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ADN/metabolismo , ARN Ligasa (ATP)/aislamiento & purificación , ARN Ligasa (ATP)/metabolismo , ARN/metabolismo , Thermus thermophilus/enzimología , Secuencia Conservada , Análisis Mutacional de ADN , ARN Ligasa (ATP)/genética , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Objective To investigate the expression and distribution of RTCB in various tissues of mice. Methods We detected the expression of RTCB by Western blotting and the location of the expression of RTCB in each tissue by immunohistochemistry. Results RTCB protein was all expressed in 17 tissues of mice. The relative expressions of oviduct, skeletal muscle and kidney were high, while the relative expressions of bladder and small intestine were low. In addition, the immunostaining of RTCB was different in different tissues or in different cells of the same tissue. The immunostaining was stronger in some specific cells of testis, epididymis, ovary, uterus, fallopian lube and small intestine. Conclusion RTCB may participate in the maintenance of reproductive function in mice.
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The activity of X box-binding protein 1 (XBP1), a master transcriptional regulator of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR), is controlled by a two-step noncanonical splicing reaction in the cytoplasm. The first step of nuclease cleavage by inositol-requiring enzyme 1 (IRE1), a protein kinase/endoribonuclease, is conserved in all eukaryotic cells. The second step of RNA ligation differs biochemically among species. In yeast, tRNA ligase 1 (Trl1) and tRNA 2'-phosphotransferase 1 (Tpt1) act through a 5'-PO4/3'-OH pathway. In metazoans, RNA 2',3'-cyclic phosphate and 5'-OH ligase (RtcB) ligate XBP1 exons via a 3'-PO4/5'-OH reaction. Although RtcB has been identified as the primary RNA ligase, evidence suggests that yeast-like ligase components may also operate in mammals. In this study, using mouse and human cell lines along with in vitro splicing assays, we investigated whether these components contribute to XBP1 splicing during ER stress. We found that the mammalian 2'-phosphotransferase Trpt1 does not contribute to XBP1 splicing even in the absence of RtcB. Instead, we found that 2',3'-cyclic nucleotide phosphodiesterase (CNP) suppresses RtcB-mediated XBP1 splicing by hydrolyzing 2',3'-cyclic phosphate into 2'-phosphate on the cleaved exon termini. By contrast, RNA 3'-terminal cyclase (RtcA), which converts 2'-phosphate back to 2',3'-cyclic phosphate, facilitated XBP1 splicing by increasing the number of compatible RNA termini for RtcB. Taken together, our results provide evidence that CNP and RtcA fine-tune XBP1 output during ER stress.
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2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Estrés del Retículo Endoplásmico/genética , Ligasas/metabolismo , Empalme del ARN , Proteína 1 de Unión a la X-Box/genética , Animales , Células HEK293 , Humanos , Ratones , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
RtcB is an essential human tRNA ligase required for ligating the 2',3'-cyclic phosphate and 5'-hydroxyl termini of cleaved tRNA halves during tRNA splicing and XBP1 fragments during endoplasmic reticulum stress. Activation of XBP1 has been implicated in various human tumors including breast cancer. Here we present, for the first time, a homology model of human RtcB (hRtcB) in complex with manganese and covalently bound GMP built from the Pyrococcus horikoshii RtcB (bRtcB) crystal structure, PDB ID 4DWQA. The structure is analyzed in terms of stereochemical quality, folding reliability, secondary structure similarity with bRtcB, druggability of the active site binding pocket and its metal-binding microenvironment. In comparison with bRtcB, loss of a manganese-coordinating water and movement of Asn226 (Asn202 in 4DWQA) to form metal-ligand coordination, demonstrates the uniqueness of the hRtcB model. Rotation of GMP leads to the formation of an additional metal-ligand coordination (Mn-O). Umbrella sampling simulations of Mn binding in wild type and the catalytically inactive C122A mutant reveal a clear reduction of Mn binding ability in the mutant, thus explaining the loss of activity therein. Our results furthermore clearly show that the GTP binding site of the enzyme is a well-defined pocket that can be utilized as target site for in silico drug discovery.
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Simulación de Dinámica Molecular , ARN Ligasa (ATP)/química , Homología de Secuencia de Aminoácido , Animales , Proteínas Bacterianas/química , Dominio Catalítico , Humanos , Manganeso/química , Manganeso/metabolismo , ARN Ligasa (ATP)/metabolismoRESUMEN
The unfolded protein response (UPR) is a conserved signalling pathway activated on the accumulation of unfolded proteins within the endoplasmic reticulum (ER), termed ER stress. Upon ER stress, HAC1/XBP1 undergoes exon/intron-specific excision by inositol requiring enzyme 1 (IRE1) to remove an intron and liberate the 5' and 3' exons. In yeast, the 5' and 3' HAC1 exons are subsequently ligated by tRNA ligase (Rlg1p), whereas XBP1 ligation in mammalian cells is catalysed by a recently identified ligase, RtcB. In the present study, RNA ligase activity of the human RtcB (hRtcB) involved in the unconventional splicing of XBP1/HAC1 mRNA was explored in an rlg1-100 mutant yeast strain. Distinct from Escherichia coli RtcB and Rlg1p, expression of hRtcB alone inefficiently complemented HAC1/XBP1 splicing and the hRtcB cofactor (archease) was required to promote enzymatic activity of hRtcB to catalyse RNA ligation.