scholarly journals Natural 2′,5′-Phosphodiester Bonds Found at the Ligation Sites of Peach Latent Mosaic Viroid

2001 ◽  
Vol 75 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Fabien Côté ◽  
Dominique Lévesque ◽  
Jean-Pierre Perreault
Keyword(s):  
Circular Rna ◽  
Rolling Circle ◽  
Phosphodiester Bonds ◽  
Biological Importance ◽  
Rolling Circle Mechanism ◽  
Ligation Site

ABSTRACT Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2′,5′-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2′,5′-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2′,5′-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2′,5′-phosphodiester bonds are still present in nature and that they are of biological importance.

scholarly journals Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two ConservedssoA Regions

1998 ◽  
Vol 180 (1) ◽  
pp. 83-89 ◽  
Author(s):  
M. Gabriela Kramer ◽  
Saleem A. Khan ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sequence Specificity ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Cell Extracts ◽  
Rolling Circle Mechanism ◽  
Derivative Plasmid ◽  
Lagging Strand

ABSTRACT The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RSB] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication inStreptococcus pneumoniae. Cells containing plasmids with mutations in the RSB accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RSB region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.

scholarly journals Monomeric Linear RNA of Citrus Exocortis Viroid Resulting from Processing In Vivo Has 5′-Phosphomonoester and 3′-Hydroxyl Termini: Implications for the RNase and RNA Ligase Involved in Replication

2008 ◽  
Vol 82 (20) ◽  
pp. 10321-10325 ◽  
Author(s):  
María-Eugenia Gas ◽  
Diego Molina-Serrano ◽  
Carmen Hernández ◽  
Ricardo Flores ◽  
José-Antonio Daròs
Keyword(s):  
Asymmetric Rolling ◽  
Rolling Circle ◽  
Rna Ligase ◽  
Citrus Exocortis Viroid ◽  
The Family ◽  
Rolling Circle Mechanism ◽  
Rapid Amplification ◽  
Hydroxyl Termini

ABSTRACT Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5′ and 3′ cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5′-phosphomonoester and 3′-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.

scholarly journals Circular RNA hsa_circ_0006401 promotes proliferation and metastasis in colorectal carcinoma

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Chenjing Zhang ◽  
Xiaolu Zhou ◽  
Xiaoge Geng ◽  
Yu Zhang ◽  
Jingya Wang ◽  
...  
Keyword(s):  
Splice Junction ◽  
Circular Rna ◽  
Host Gene ◽  
Cell Counting ◽  
Tissue Samples ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Proliferation And Migration

AbstractDysregulation of circular RNA (circRNA) expression is involved in the progression of cancer. Here, we aimed to study the potential function of hsa_circ_0006401 in colorectal cancer (CRC). CircRNA hsa_circ_0006401 expression levels in CRC and adjacent nontumor tissues were analyzed by real-time quantitative PCR (qRT-PCR) and circRNA in situ hybridization (RNA-ISH). Then, CRC cell proliferation was assessed by cell counting. Wound-healing and transwell assays were utilized to detect the effect of hsa_circ_0006401 on CRC migration. A circRNA-ORF construct was created, and a specific antibody against the splice junction of hsa_circ_0006401 was prepared. Finally, the proteins directly binding to hsa_circ_0006401 peptides were identified by immunoprecipitation combined with mass spectrometry. In our study, we found hsa_circ_0006401 was closely related to CRC metastasis and exhibited upregulated expression in metastatic CRC tissue samples. Proliferation and migration were inhibited in vitro when hsa_circ_0006401 expression was silenced. Downregulation of hsa_circ_0006401 expression decreased CRC proliferation and liver metastasis in vivo. A 198-aa peptide was encoded by sequences of the splice junction absent from col6a3. Hsa_circ_0006401 promoted CRC proliferation and migration by encoding the hsa_circ_0006401 peptide. Hsa_circ_0006401 peptides decreased the mRNA and protein level of the host gene col6a3 by promoting col6a3 mRNA stabilation. In conclusion, our study revealed that circRNAs generated from col6a3 that contain an open-reading frame (ORF) encode a novel 198-aa functional peptide and hsa_circ_0006401 peptides promote stability of the host gene col6a3 mRNA to promote CRC proliferation and metastasis.

scholarly journals CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Junjie Xu ◽  
Lin Ji ◽  
Yuelong Liang ◽  
Zhe Wan ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Nuclear Interaction ◽  
Circular Rna ◽  
Advanced Hepatocellular Carcinoma ◽  
Proof Of Concept ◽  
Potential Strategy ◽  
Oncogenic Protein ◽  
Hcc Cells

AbstractSorafenib is the first-line chemotherapeutic therapy for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance significantly limits its therapeutic efficacy, and the mechanisms underlying resistance have not been fully clarified. Here we report that a circular RNA, circRNA-SORE (a circular RNA upregulated in sorafenib-resistant HCC cells), plays a significant role in sorafenib resistance in HCC. We found that circRNA-SORE is upregulated in sorafenib-resistant HCC cells and depletion of circRNA-SORE substantially increases the cell-killing ability of sorafenib. Further studies revealed that circRNA-SORE binds the master oncogenic protein YBX1 in the cytoplasm, which prevents YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and thus blocks PRP19-mediated YBX1 degradation. Moreover, our in vitro and in vivo results suggest that circRNA-SORE is transported by exosomes to spread sorafenib resistance among HCC cells. Using different HCC mouse models, we demonstrated that silencing circRNA-SORE by injection of siRNA could substantially overcome sorafenib resistance. Our study provides a proof-of-concept demonstration for a potential strategy to overcome sorafenib resistance in HCC patients by targeting circRNA-SORE or YBX1.

scholarly journals Circular RNA CircPPP1CB Suppresses Tumorigenesis by Interacting With the MiR-1307-3p/SMG1 Axis in Human Bladder Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Feifan Wang ◽  
Yan Zhang ◽  
Xuejian Zhou ◽  
Xianwu Chen ◽  
Jiayong Xiang ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Epithelial To Mesenchymal Transition ◽  
Human Cancer ◽  
Circular Rna ◽  
Human Bladder Cancer ◽  
Human Bladder ◽  
Mesenchymal Transition ◽  
Circular Structure

Circular RNA (circRNA) is a newly discovered endogenous non-coding RNA (ncRNA), which is characterized with a closed circular structure. A growing body of evidence has verified the vital roles of circRNAs in human cancer. In this research, we selected circPPP1CB as a study object by circRNA sequencing and quantitative real-time PCR (qRT-PCR) validation in human bladder cancer (BC). CircPPP1CB is downregulated in BC and is negatively correlated with clinical stages and histological grades. Functionally, circPPP1CB modulated cell growth, metastasis, and epithelial-to-mesenchymal transition (EMT) process in vitro and in vivo. Mechanically, we performed various experiments to verify the circPPP1CB/miR-1307-3p/SMG1 regulatory axis. Taken together, our results demonstrated that circPPP1CB participates in tumor growth, metastasis, and EMT process by interacting with the miR-1307-3p/SMG1 axis, and that circPPP1CB might be a novel therapeutic target and diagnostic biomarker in human BC.

scholarly journals Circular RNA CDR1as Inhibits the Metastasis of Gastric Cancer through Targeting miR-876-5p/GNG7 Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jiajia Jiang ◽  
Rong Li ◽  
Junyi Wang ◽  
Jie Hou ◽  
Hui Qian ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Underlying Mechanisms

Circular RNA CDR1as has been demonstrated to participate in various cancer progressions as miRNA sponges. The exact underlying mechanisms of CDR1as on gastric cancer (GC) metastasis remain unknown. Here, we found that CDR1as knockdown facilitated GC cell migration and invasion while its overexpression inhibited the migration and invasion abilities of GC cells in vitro and in vivo. Moreover, epithelial-mesenchymal transition- (EMT-) associated proteins and MMP2 and MMP9 were downregulated by CDR1as. Bioinformatics analysis combined with dual-luciferase reporter gene assays, western blot, RT-qPCR analysis, and functional rescue experiments demonstrated that CDR1as served as a miR-876-5p sponge and upregulated the target gene GNG7 expression to suppress GC metastasis. In summary, our findings indicate that CDR1as suppresses GC metastasis through the CDR1as/miR-876-5p/GNG7 axis.

scholarly journals circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Zhecheng Wang ◽  
Yan Zhao ◽  
Ruimin Sun ◽  
Yu Sun ◽  
Deshun Liu ◽  
...  
Keyword(s):  
Liver Injury ◽  
Mitochondrial Dynamics ◽  
Circular Rna ◽  
Protective Effects ◽  
Bioinformatics Analyses ◽  
Hepatocyte Injury ◽  
Rna Immunoprecipitation ◽  
Competitive Endogenous Rna

Abstract p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.

scholarly journals Circular RNA CDR1as Exerts Oncogenic Properties Partially through Regulating MicroRNA 641 in Cholangiocarcinoma

2020 ◽  
Vol 40 (15) ◽  
Author(s):  
Dingyang Li ◽  
Zhe Tang ◽  
Zhiqiang Gao ◽  
Pengcheng Shen ◽  
Zhaochen Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Target Genes ◽  
Circular Rna ◽  
Tumor Xenograft ◽  
Mrna Levels ◽  
Cell Behaviors ◽  
Xenograft Growth ◽  
Tumor Xenograft Growth

ABSTRACT It has been found that the circular RNA (circRNA) CDR1as is upregulated in cholangiocarcinoma (CCA) tissues. In this study, we tried to explore the roles of CDR1as in CCA. CDR1as was overexpressed or knocked down in human CCA cells to assess the effects of CDR1as on cell behaviors and tumor xenograft growth. In vitro, the CDR1as level was significantly increased in CCA cell lines. The results showed that CDR1as promoted the cell proliferation, migration, invasion, and activation of the AKT3/mTOR pathway in CCA cells. Moreover, miR-641, a predicted target microRNA (miRNA) of CDR1as, could partially reverse the effects of CDR1as on cell behaviors in CCA cells. Furthermore, CDR1as improved tumor xenograft growth, and it could be attenuated by miR-641 in vivo. Additionally, CDR1as expression was inversely correlated with miR-641 in CCA cells, and miR-641 could directly bind with CDR1as and its target genes, the AKT3 and mTOR genes. Mechanistically, CDR1as could bind with miR-641 and accelerate miR-641 degradation, which possibly leads to the upregulation of the relative mRNA levels of AKT3 and mTOR in RBE cells. In conclusion, our findings indicated that CDR1as might exert oncogenic properties, at least partially, by regulating miR-641 in CCA. CDR1as and miR-641 could be considered therapeutic targets for CCA.

scholarly journals Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo

2000 ◽  
Vol 74 (20) ◽  
pp. 9451-9463 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Theresa A. Storm ◽  
Mark A. Kay
Keyword(s):  
Rolling Circle Replication ◽  
Chromosomal Dna ◽  
Adeno Associated Virus ◽  
Rolling Circle ◽  
Steady State Levels ◽  
Slow Rise ◽  
Mouse Hepatocytes ◽  
Enzyme Recognition

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

scholarly journals CircSMAD4 alleviates high glucose-induced inflammation, extracellular matrix deposition and apoptosis in mouse glomerulus mesangial cells by relieving miR-377-3p-mediated BMP7 inhibition

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Rina Wu ◽  
Zheli Niu ◽  
Guangwei Ren ◽  
Lin Ruan ◽  
Lijun Sun
Keyword(s):  
Extracellular Matrix ◽  
High Glucose ◽  
Mesangial Cells ◽  
Circular Rna ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Matrix Deposition

Abstract Background Diabetic nephropathy (DN) is a common complication of diabetes mellitus. Accumulating studies suggest that the deregulation of circular RNA (circRNA) is involved in DN pathogenesis. This study aimed to investigate the role of circSMAD4 in DN models. Methods Mice were treated with streptozotocin to establish DN models in vivo. Mouse glomerulus mesangial cells (SV40-MES13) were treated with high glucose to establish DN models in vitro. The expression of circSMAD4, miR-377-3p and bone morphogenetic protein 7 (BMP7) mRNA was measured by quantitative real-time PCR (qPCR). The releases of inflammatory factors were examined by ELISA. The protein levels of fibrosis-related markers, apoptosis-related markers and BMP7 were checked by western blot. Cell apoptosis was monitored by flow cytometry assay. The predicted relationship between miR-377-3p and circSMAD4 or BMP7 was validated by dual-luciferase reporter assay or pull-down assay. Results CircSMAD4 was poorly expressed in DN mice and HG-treated SV40-MES13 cells. HG induced SV40-MES13 cell inflammation, extracellular matrix (ECM) deposition and apoptosis. CircSMAD4 overexpression alleviated, while circSMAD4 knockdown aggravated HG-induced SV40-MES13 cell injuries. MiR-377-3p was targeted by circSMAD4, and miR-377-3p enrichment partly reversed the effects of circSMAD4 overexpression. BMP7 was a target of miR-377-3p, and circSMAD4 regulated BMP7 expression by targeting miR-377-3p. MiR-377-3p overexpression aggravated HG-induced injuries by suppressing BMP7. Conclusion CircSMAD4 alleviates HG-induced SV40-MES13 cell inflammation, ECM deposition and apoptosis by relieving miR-377-3p-mediated inhibition on BMP7 in DN progression.

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