scholarly journals The 3’tsRNAs are aminoacylated: Implications for their biogenesis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (7) ◽  
pp. e1009675
Author(s):  
Ziwei Liu ◽  
Hak Kyun Kim ◽  
Jianpeng Xu ◽  
Yuqing Jing ◽  
Mark A. Kay
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Small Rnas ◽  
Ribosome Biogenesis ◽  
Trna Synthetase ◽  
Fine Tuning ◽  
Enzymatic Method ◽  
Dividing Cells ◽  
Transcriptional Gene Regulation ◽  
Induced Apoptosis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG 3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG 3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.

scholarly journals The 3'tsRNAs are aminoacylated: Implications for their biogenesis

2020 ◽  
Author(s):  
Ziwei Liu ◽  
Hak kyun Kim ◽  
Jianpeng Xu ◽  
Mark A Kay
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Small Rnas ◽  
Ribosome Biogenesis ◽  
Trna Synthetase ◽  
Fine Tuning ◽  
Enzymatic Method ◽  
Dividing Cells ◽  
Transcriptional Gene Regulation ◽  
Induced Apoptosis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from 3' end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3'-tsRNAs remains elusive and it is unclear if the 3'-ends of 3'-tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3'charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3'-tsRNA is fully charged and originated solely from charged mature tRNA. When the leucyl-tRNA synthetase was knocked down, less tsRNA was generated while the mature tRNA was not reduced further supporting that tsRNA generation is regulated. The fact that the 3'-tsRNA is aminoacylated has implications for their biogenesis and provides additional insights into their biological role in post-transcriptional gene regulation.

scholarly journals Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
René F. Ketting
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Gene Silencing ◽  
Small Rnas ◽  
Self Regulation ◽  
Fine Tuning ◽  
Specific Gene ◽  
Next Generation ◽  
C Elegans ◽  
Argonaute Proteins

AbstractEndogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we find that sRNA abundance, the pattern of origin of sRNA and 3’ UTR length are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we discovered that ALG-3- and ALG-4-bound 26G-RNAs are dampening the expression of their own mRNAs, revealing a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.Author SummarySmall RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are important to initiate specific gene expression programs in the next generation. The nematode Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs that are divided into two subpopulations: one expressed in the spermatogenic gonad and another expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression.

Effects of 5-aza-2ˈ-deoxycytidine and Valproic Acid on Epigenetic-modifying DNMT1 Gene Expression, Apoptosis Induction and Cell Viability in Hepatocellular Carcinoma WCH-17 cell line

2019 ◽  
Author(s):  
Masumeh Sanaei ◽  
Fraidoon Kavoosi
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Valproic Acid ◽  
Cell Growth ◽  
Cell Line ◽  
Dna Methyltransferase ◽  
Transcriptional Control ◽  
Treatment Time ◽  
Induced Apoptosis ◽  
Apoptotic Effects

Background: DNA molecule of the eukaryotic cells is found in the form of a nucleoprotein complex named chromatin. Two epigenetic modifications are critical for transcriptional control of genes, including acetylation and DNA methylation. Hypermethylation of tumor suppressor genes is catalyzed by various DNA methyltransferase enzymes (DNMTs), including DNMT1, DNMT2, and DNMT3. The most well characterized DNA demetilating and histone deacetylase inhibitor drugs are 5-aza-2ˈ-deoxycytidine (5-Aza-CdR) and valproic acid (VPA), respectively. The purpose of the current study was to analyze the effects of 5-Aza-CdR and VPA on cell growth, apoptosis, and DNMT1 gene expression in the WCH-17 hepatocellular carcinoma (HCC) cell line. Materials and Methods: In this descriptive analytical study, MTT assay, flow cytometry assay, and Quantitative Real-Time RT-PCRwere done to evaluate proliferative and apoptotic effects and also gene expression. Results: Both compounds inhibited the cell growth and induced apoptosis significantly in a dose and time depended fashion. Additionally, 5-Aza-CdR down-regulated DNMT1 gene expression. The relative expression of DNMT1 was 0.40 and 0.20 (P < 0.001) at different times, respectively. The percentage of VPA- treated apoptotic cells were reduced by about 28 and 34 % (P˂0.001) and that of 5-Aza-CdR-treated were reduced by about 34 and 44 % (P˂0.001) after treatment time periods. Conclusion: In the current study, it was observed that 5-Aza-CdR and VPA could significantly inhibit the growth of WCH-17 cell and played a significant role in apoptosis. It was also found that 5-Aza-CdR could decrease DNMT1 gene expression.

scholarly journals Vibrio choleraeCsrA Directly RegulatesvarATo Increase Expression of the Three Nonredundant Csr Small RNAs

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Heidi A. Butz ◽  
Alexandra R. Mey ◽  
Ashley L. Ciosek ◽  
Shelley M. Payne
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Small Rnas ◽  
Feedback Loop ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Fine Tuning ◽  
Mobility Shift ◽  
Content Type

ABSTRACTCsrA, an RNA-binding global regulator, is an essential protein inVibrio cholerae.V. choleraeCsrA is regulated by three small RNAs (sRNAs), namely, CsrB, CsrC, and CsrD, which act to sequester and antagonize the activity of CsrA. Although the sRNAs were considered to be largely redundant, we found that they differ in expression, half-life, and the ability to regulate CsrA. Further, we identified a feedback loop in the Csr system in which CsrA increases the synthesis of these antagonistic sRNAs. Because the Csr sRNAs are positively regulated by VarA, we determined the effects of CsrA on VarA levels. The level of VarA was reduced in acsrAmutant, and we found that CsrA directly bound tovarAmRNA in an electrophoretic mobility shift assayin vitroand in an CsrA-RNA immunoprecipitation assayin vivo. Thus,varAmRNA is anin vivo-verified direct target of CsrA inV. cholerae, and this is the first demonstration of CsrA directly binding to avarA/uvrY/gacAhomolog. Additionally, we demonstrated that avarAtranslational fusion was less active in acsrAmutant than in wild-typeV. cholerae, suggesting that CsrA enhancesvarAtranslation. We propose that this autoregulatory feedback loop, in which CsrA increases the production of the nonredundant Csr sRNAs by regulating the amount of VarA, provides a mechanism for fine-tuning the availability of CsrA and, thus, of its downstream targets.IMPORTANCEVibrio choleraeis a major human pathogen, causing epidemics and pandemics of cholera.V. choleraepersists in the aquatic environment, providing a constant source for human infection. Success in transitioning from the environment to the human host and back requires the bacterium to rapidly respond and to adjust its gene expression and metabolism to these two very different habitats. Our findings show that CsrA, an RNA-binding regulatory protein, plays a central role in regulating these transitions. CsrA activity is controlled by the antagonistic sRNAs CsrB, CsrC, and CsrD, and these sRNAs respond to changes in the availability of nutrients. CsrA autoregulates its own activity by controlling these sRNAs via their primary regulator VarA. Thus, the change in CsrA availability in response to nutrient availability allowsV. choleraeto alter gene expression in response to environmental cues.

Synergistic Effect of α-Solanine and Cisplatin Induces Apoptosis and Enhances Cell Cycle Arrest in Human Hepatocellular Carcinoma Cells

2020 ◽  
Vol 19 (18) ◽  
pp. 2197-2210 ◽  
Author(s):  
Sherien M. El-Daly ◽  
Shaimaa A. Gouhar ◽  
Amira M. Gamal-Eldeen ◽  
Fatma F. Abdel Hamid ◽  
Magdi N. Ashour ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Synergistic Effect ◽  
Cell Cycle Arrest ◽  
Combination Treatment ◽  
Carcinoma Cells ◽  
Cell Growth Inhibition ◽  
Cycle Arrest ◽  
Induced Apoptosis ◽  
Human Hepatocellular Carcinoma Cells

Aim: The clinical application of cisplatin is limited by severe side effects associated with high applied doses. The synergistic effect of a combination treatment of a low dose of cisplatin with the natural alkaloid α-solanine on human hepatocellular carcinoma cells was evaluated. Methods: HepG2 cells were exposed to low doses of α-solanine and cisplatin, either independently or in combination. The efficiency of this treatment modality was evaluated by investigating cell growth inhibition, cell cycle arrest, and apoptosis enhancement. Results: α-solanine synergistically potentiated the effect of cisplatin on cell growth inhibition and significantly induced apoptosis. This synergistic effect was mediated by inducing cell cycle arrest at the G2/M phase, enhancing DNA fragmentation and increasing apoptosis through the activation of caspase 3/7 and/or elevating the expression of the death receptors DR4 and DR5. The induced apoptosis from this combination treatment was also mediated by reducing the expression of the anti-apoptotic mediators Bcl-2 and survivin, as well as by modulating the miR-21 expression. Conclusion: Our study provides strong evidence that a combination treatment of low doses of α-solanine and cisplatin exerts a synergistic anticancer effect and provides an effective treatment strategy against hepatocellular carcinoma.

Synergistic Antitumor Effect of 5-Fluorouracil Combined with Constituents from Pleurospermum lindleyanum in Hepatocellular carcinoma SMMC-7721 Cells

2020 ◽  
Vol 20 ◽  
Author(s):  
Xiao-Feng Zhu ◽  
Xiao-Jin Li ◽  
Zhong-Lian Cao ◽  
Xiu-Jie Liu ◽  
Ping Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Treatment Effectiveness ◽  
Synergistic Effects ◽  
Folk Medicine ◽  
Inhibitory Effect ◽  
Caspase 9 ◽  
Whole Plant ◽  
Anti Cancer ◽  
Induced Apoptosis

Background: A Chinese folk medicine plant Pleurospermum lindleyanum possesses pharmacological activities of heat-clearing, detoxifying and preventing from hepatopathy, coronary heart disease, hypertension, and high altitude sickness. We isolated and characterized its constituents to investigate its synergistic effects against human hepatoma SMMC-7721 cells. Objective: The aim of this study was to explore the synergistic anti-cancer activities of isolates from P. lindleyanum with 5-FU on hepatoma SMMC-7721 cells in vitro and their primary mechanisms. Methods: Sequential chromatographic techniques were conducted for the isolation studies. The isolates structures were established by spectroscopic analysis as well as X-ray crystallographic diffraction. Growth inhibition was detected by MTT assay. The isobologram method was used to assess the effect of drug combinations. Flow cytometry and western blot were used to examine apoptosis and protein expression. Results: A new coumarin (16), along with sixteen known compounds, were isolated from the whole plant of P. lindleyanum and their structures were elucidated by spectroscopic methods. Four coumarins (2, 3, 5, and 16), two flavonoids (8 and 9) and three phytosterols and triterpenes (12-14) were found to synergistically enhance the inhibitory effect of 5-FU against SMMC-7721 cells. Among them, compounds 3 and 16 exhibited the best synergistic effects with IC50 of 5-FU reduced by 16-fold and 22-fold possessing the minimum Combination Index (CI) 0.34 and 0.27. The mechanism of action of combinations might be through synergistic arresting for the cell cycle at G1 phases and the induction of apoptosis. Moreover, western blotting and molecular docking revealed that compounds 3 or 5 might promote 5-FU-induced apoptosis by regulating the expression of Caspase 9 and PARP. Conclusion: Constituents from P. lindleyanum may improve the treatment effectiveness of 5-FU against hepatocellular carcinoma cells.

Hepatocellular carcinoma: Gene expression profiling and regulation of xenobiotic-metabolizing cytochromes P450

2020 ◽  
Vol 177 ◽  
pp. 113912 ◽  
Author(s):  
Jana Nekvindova ◽  
Alena Mrkvicova ◽  
Veronika Zubanova ◽  
Alena Hyrslova Vaculova ◽  
Pavel Anzenbacher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Cytochromes P450
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