• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.6
• /
• pp.812-819
• /
• 2018

Objective: An experiment was conducted to identify and characterize the circular RNA expression and metabolic characteristics in the liver of Jinhua pigs and Landrace pigs. Methods: Three Jinhua pigs and three Landrace pigs respectively at 70-day were slaughtered to collect the liver tissue samples. Immediately after slaughter, blood samples were taken to detect serum biochemical indicators. Total RNA extracted from liver tissue samples were used to prepare the library and then sequence on HiSeq 2500. Bioinformatic methods were employed to analyze sequence data to identify the circRNAs and predict the potential roles of differentially expressed circRNAs between the two breeds. Results: Significant differences in physiological and biochemical traits were observed between growing Jinhua and Landrace pigs. We identified 84,864 circRNA candidates in two breeds and 366 circRNAs were detected as significantly differentially expressed. Their host genes are involved in lipid biosynthetic and metabolic processes according to the gene ontology analysis and associated with metabolic pathways. Conclusion: Our research represents the first description of circRNA profiles in the porcine liver from two divergent phenotype pigs. The predicted miRNA-circRNA interaction provides important basis for miRNA-circRNA relationships in the porcine liver. These data expand the repertories of porcine circRNA and are conducive to understanding the possible molecular mechanisms involved in miRNA and circRNA. Our study provides basic data for further research of the biological functions of circRNAs in the porcine liver.

• Korean Journal of Microbiology
• /
• v.47 no.4
• /
• pp.289-294
• /
• 2011

We investigated the cold shock sensitivity of DEAD-box RNA helicase gene deleted strains of in Bacillus subtilis CU1065. To understand cold shock effects, cells were cultivated at $37^{\circ}C$ to log phase ($O.D_{600}$=0.5-0.6) and then temperature was shifted to $15^{\circ}C$. Cold shock slow down the growth rate of wild type and deleted strains of DEAD-box RNA helicase gene (ydbR, yfmL, yqfR, deaD). The growth rate of ydbR deleted strain is 5 times severely reduced compared to that of wild type strain (CU1065). But the growth rate of other three (yfmL, yqfR, deaD) deleted strains is nearly equal to the growth rate of wild type. Compared to $37^{\circ}C$, the amount of ydbR and yqfR mRNA transcripts are increased at the growth temperature of $15^{\circ}C$. On the other hands the mRNA transcripts of yfmL and deaD are not changed at both conditions of $37^{\circ}C$ and $15^{\circ}C$. Upon cold shock treatment ydbR mRNA transcript is clearly increased. After treatment of rifampicin (bacteria transcription inhibitor) the amount of ydbR mRNA was measured. Temperature shift from $37^{\circ}C$ to $15^{\circ}C$ and rifampicin treatment showed slowly decay of ydbR mRNA. But at $37^{\circ}C$ and rifampicin treatment ydbR mRNA is rapidly reduced. These results showed that cold shock induction of ydbR mRNA resulted from the stability of ydbR mRNA and not from the transcription induction of ydbR. In relation to these results, we found the cold box element of csp (cold shock protein gene) in 5' untranslated region of ydbR gene. Cold shock induction of ydbR is caused by the stability of ydbR mRNA like the stability of csp mRNA.

• Animal Bioscience
• /
• v.36 no.4
• /
• pp.555-569
• /
• 2023

Objective: The objective of this study was to investigate the effects of N⁶-Methyladenosine modification-circRNA-zinc finger protein 638 (m⁶A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats. Methods: The m⁶A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m⁶A dependence were evaluated through the overexpression of circRNA-ZNF638/its m⁶A-deficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3'- untranslated region was analyzed through Dual-luciferase reporter assay. Results: The m⁶A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m⁶A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHF-stem cells. We further demonstrated that the internal m⁶A modification within circRNA-ZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m⁶A-deficient mutant of circRNA-ZNF638. Conclusion: The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m⁶A-dependent manner through miR-361-5p/Wnt5a axis.

• Korean Journal Plant Pathology
• /
• v.3 no.4
• /
• pp.239-244
• /
• 1987

Low molecular weight plant ribonucleic acids including potato spindle tuber viroid(PSTV) RNA were electrophoresed in 0M to 8M urea-gradient polyacrylamide gels. The electrophoresis was carried on in a urea - gradient gel system with 1/40 and 1/10 dilution of TBE buffer at three different temperatures, $17^{\circ}C,\;37^{\circ}C\;and\;57^{\circ}C$. The most effective separation of PSTV - RNA molecules into circular and linear forms was achieved at the highly denaturing temperature of $57^{\circ}C$ and at 1/40 dilution of TBE buffer. The electrophoretic mobility of the denatured circular viroid-RNA molecules is dependent mainly on the concentration of urea. In addition, a low concentration of TBE buffer would increase the separation distance between the circular and linear forms of PSTV-RNA molecules in the denaturing urea-gradient gel system

• Korean Journal of Environmental Biology
• /
• v.33 no.4
• /
• pp.433-440
• /
• 2015

The Apostichopus japonicus is an important species in some Asia countries including Korea, China and Japan. The purpose of the present study was to investigate the differential gene expression of heat shock protein90 (Hsp90) and ferritin as a biomarker for the thermal stress during water temperature rising in the sea cucumber, A. japonicus. The A. japonicus (1.4 g) was cultured in incubator of separate temperature ($15^{\circ}C$, $20^{\circ}C$, $25^{\circ}C$ and $30^{\circ}C$) for each 0, 3, 6, 12, 24, 48 hours. The mRNA expression levels of Hsp90 and ferritin were examined using RT-PCR assay. Results showed that, the expression of Hsp90 mRNA was not significantly changed at $15^{\circ}C$. The expression of Hsp90 mRNA was significantly increased at high temperature such as $20^{\circ}C$ and $25^{\circ}C$. Furthermore, Hsp90 mRNA was early increased at $25^{\circ}C$ than $20^{\circ}C$. The ferritin mRNA was similar expression pattern with Hsp90. But, Hsp90 mRNA was more sensitive than ferritin mRNA at high thermal stress. These results indicate that Hsp90 and ferritin mRNAs were involved in the temperature changes response and may be play an important role in mediating the thermal stress in A. japonicas.

• Applied Biological Chemistry
• /
• v.38 no.6
• /
• pp.522-527
• /
• 1995

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with $T_3$ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at $55^{\circ}C$, while the cleavage was not detected at $37^{\circ}C$. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at $55^{\circ}C$. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.

• Journal of Gastric Cancer
• /
• v.20 no.3
• /
• pp.300-312
• /
• 2020

Purpose: Circular RNAs (circRNAs) are a new class of RNA molecules whose function is largely unknown. There is a growing evidence that circRNAs play an important regulatory role in the progression of a variety of human cancers. However, the exact roles and the mechanisms of circRNAs in gastric cancer are not clear. In this study, we aimed to elucidate the mechanism of hsa_circ_0005556. Materials and Methods: Real-time quantitative polymerase chain reaction was used to detect the expression of hsa_circ_0005556, miR-4270, and matrix metalloproteinase-19 (MMP19) in gastric cancer tissues and cell lines. The expression of hsa_circ_0005556 in gastric cancer cells was silenced by lentivirus, and cell proliferation, invasion, migration, and tumorigenesis in nude mice were assessed to evaluate the function of hsa_circ_0005556 in gastric cancer. Results: The expression of hsa_circ_0005556 in gastric cancer tissues and gastric cancer cell lines was higher compared to normal controls. In vitro, the downregulation of hsa_ circ_0005556 significantly inhibited proliferation, migration, and invasion of gastric cancer cells. In vivo, the downregulation of hsa_circ_0005556 suppressed tumor growth in nude mice. Conclusions: Our study shows that the hsa_circ_0005556/miR-4270/MMP19 axis is involved in proliferation, migration, and invasion of gastric cancer cells through the competing endogenous RNA (ceRNA) mechanism.

• Applied Biological Chemistry
• /
• v.37 no.1
• /
• pp.56-63
• /
• 1994

Oligonucleotides for a conserved region of the coat protein gene of cucumber mosaic virus (CMV) and a hammerhead structure ribozyme against CMV RNA were synthesized using a DNA synthesizer. Both strands of oligonucleotides were annealed and restricted with BamHI/SacI, then cloned into a plasmid pBS SK (+). The cloned CMV substrate and ribozyme were sequenced to verify correct constructions. In vitro transcriptions were carried out by using T7 RNA polymerase with BssHII or SspI digests of $1\;{\mu}g$ of substrate and ribozyme clones. The size of substrate RNA was 176 nucleotides (nt) containing 50 nt of CMV RNA sequence, 6 nt of XbaI restriction site and 120 nt of vector-derived sequence in the case of BssHII digest. The size of ribozyme RNA was 164 nt containing 40 nt of ribozyme RNA sequence and same sequences of substrate. Substrate RNA was efficiently cleaved into two fragments (96 nt and 80 nt) by ribozyme RNA. This endonucleolytic cleavage occurred more efficiently at $55^{\circ}C$ than $37^{\circ}C$. SspI digest-derived substrate RNA (2234 nt) was also cleaved into two fragments by the same ribozyme. SspI digest-derived ribozyme RNA (2222 nt) cleaved the above substrate to two fragments. In vitro-tested ribozyme construct is being cloned into a plant transformation vector to develop virus-resistant plants.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.10
• /
• pp.1550-1557
• /
• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• Korean Journal of Food Science and Technology
• /
• v.27 no.1
• /
• pp.129-133
• /
• 1995

Continuous fermentations were performed in order to investigate the effect of culture condition on the yeast RNA accumulation and autolysis efficiency. The content of intracellular RNA increased with increasing dilution rate, showing its maximum value of 14.8% at D=0.35 $h^{-1}$. Also, both RNA productivity and specific RNA productivity tended to increase with the increase of dilution rate. The maximum biomass was obtained at $30^{\circ}C$ in the fixed dilution rate of 0.2 $h^{-1}$, whereas the maximum RNA content appeared at the lowest temperature experimented. Growth rate affected significantly on the yeast autolysis efficiency such that the extraction ratio(TN/TN) increased with increasing growth rate, whereas the hydrolysis ratio(AN/TN) was reversed. On the other hand, its efficiency was little affected by cultivation temperature.