• Journal of the Korean Chemical Society
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• v.39 no.6
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• pp.453-458
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• 1995

We have applied Pb2+ to the structural analysis of 5S rRNA from Pseudomonas alcaligenes. The mode of Pb2+-induced clevage on 5S rRNA has shown several specific features which may be utilized for the examination of tertiary structure of 5S rRNA: Pb2+ does not attack the stable helical stems; single stranded regions or bulges are attacked in variable susceptibilities depending on the positions of the sequences or the bases on the molecule; unstable helical region d is not attacked at all; only 3' sided strand of unstable helical stem C is weakly attacked, leaving 5' sided strand unattacked. Based on the Pb2+ cleavage properties and the structural analysis of Xanthomonas celebensis 5S rRNA, we have proposed a working hypothesis for the tertiary interactions in 5S rRNA.

• BMB Reports
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• v.57 no.1
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• pp.40-49
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• 2024

Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNA-guided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies.

• Proceedings of the Korean Information Science Society Conference
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• 2003.10b
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• pp.826-828
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• 2003

이제까지 Protein이나 RNA와 같은 분자의 구조는, 대부분 X-ray crystallography나 Nuclear Magnetic Resonance (NMR) 방법을 통해 분석이 이루어 졌다. 이 방법들은 실제 분자를 직접 원자레벨에서 분석하는 방법으로, 분자를 구성하는 모든 원자의 3차원 좌표 정보를 얻어 낼 수 있다. 원자의 3차원 좌표 정보는 분자의 전체적인 모양과 구조를 이해하는데 유용한 정보이다. 하지만, 분자의 구조를 좀 더 완벽히 이해하기 위해서는 원자 레벨의 좌표 정보 보다는 좀 더 높은 차원에서의 구조 정보가 필요하다. 특히 분자의 구조를 예측하거나, 분자들 사이에 결합 관계를 예측하기 위해서는, 원자 레벨의 정보만으로는 필요한 모든 정보를 얻을 수 없다. 이러한 경우, 분자의 2차원 또는 3차원 구조 요소 (structural elements)가 더욱 좋은 정보를 제공해 줄 수 있다. Protein 분자의 경우. 이미 3차원 좌표 정보를 이용해서, 2차원 구조 요소를 알아내는 자동화된 방법이 알려져 있다. 그러나 RNA의 경우 protein에 비해 알려진 결정 구조가 적기 때문에. 아직까지 2차원 구조 요소나 3차원 구조 요소를 알아내는 자동화된 방법이 알려져 있지 않다. 따라서, 이제까지는 RNA의 구조 요소를 알아내기 위해, 사람이 직접 RNA분자의 3차원 좌표 정보를 분석함으로써 많은 시간과 노력이 필요했다. 이 때문에, 우리는 RNA의 원자들의 3차원 좌표 정보를 이용해서, 2차원 구조요소와 3차원 구조 요소 정보를 자동화된 방법으로 밝혀내는 알고리즘을 개발하였다. 우리는 분자를 구성하고 있는 원자들의 3차원 좌표 정보를 Protein data bank (PDB)에서 가져왔다. 우리의 알고리즘은 PDB file형태의 데이터라면 protein-RNA 복합체나 RNA 분자 모두에서 RNA의 2차원 구조 요소나 3차원 구조 요소를 얻어낼 수 있다. 우리의 연구는 RNA의 원자레벨의 3차원 좌표 정보를 이용해서 RNA의 구조 요소를 뽑아내는 첫 번째 시도로, 우리의 알고리즘을 통해 얻어진 구조 정보는 RNA의 구조 예측 연구나. protein-RNA complex의 결합 예측 연구에 많은 도움을 줄 수 있으리라 기대된다.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.15-20
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• 2010

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.3
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.2
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• pp.46-49
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• 2016

MiRNA-155, upregulated in various cancers, is one of the miRNAs that suppress apoptosis of human cancer. Thus, inhibition of the maturation of miRNA-155 could be an effective way to induce apoptotic cancer cell death. The apical stem-loop of the pre-miRNA-155 has been known as a Dicer biding site for RNA cleavage. Here, to understand the molecular basis of the tertiary interaction between pre-miRNA-155 with Dicer, we characterize the structure of the apical stem-loop of pre-miRNA-155 using NMR spectroscopy. The RNA has a stem-bulge-stem-loop-stem structure, which is consist of G-C Watson-Crick and G-U Wobble base pairs. The assignments of imino- protons were further confirmed by 2D $^{15}N-^1H$ HSQC NMR spectrum. The NMR parameters obtained in this study can be further used to investigate the tertiary interaction between pre-miRNA-155 and other biomolecules such as protein, nucleic acids, or small chemicals which might be used to control the apoptosis of cancer.

• The Plant Pathology Journal
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• v.27 no.3
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• pp.285-290
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• 2011

Fusarium graminearum virus 2 (FgV2) infects Fusarium graminearum strain 98-8-60 and has at least five segments of double-stranded RNAs (dsRNAs), denoted as dsRNA-1 to dsRNA-5. In this study, the genome of FgV2 was sequenced and its phylogenetic relationship with other mycoviruses was analyzed. The lengths of FgV2 dsRNAs 1-5 ranged from 2414 to 3580 base pairs (bp). The 5' and 3' untranslated regions (UTRs) are highly conserved, and each dsRNA segment had 78-105 and 84-306 bp of 5' and 3' UTRs, respectively. Each dsRNA segment contained a single open reading frame (ORF). Computer analysis of dsRNA-1 revealed a putative open reading frame (ORF) that shows high sequence identity with an RNA-dependent RNA polymerase (RdRp) containing eight conserved motifs. dsRNAs 2-5 also each contain one putative ORF coding for products of unknown function. The sequences of FgV2 dsRNA-2 and dsRNA-3 have significant sequence identity with Magnaporthe oryzae chrysovirus 1 (MoCV1) dsRNA-3 and -4, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis of the putative RdRp protein, FgV2 was found to form a distinct virus clade with Aspergillus mycovirus 1816 and MoCV1 in the family Chrysoviridae.

• Molecules and Cells
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• v.26 no.3
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• pp.308-313
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• 2008

In animals, microRNAs (miRNAs) and small interfering RNAs (siRNAs) repress expression of protein coding genes by assembling distinct RNA-induced silencing complexes (RISCs). It has previously been shown that passenger-strand cleavage is the predominant mechanism when siRNA duplexes are loaded into Argonaute2 (Ago2)-containing RISC, while an unwinding bypass mechanism is favored for miRNA duplexes with mismatches. Here I present experimental data indicating that some mammalian miRNAs are assembled into Ago2-containing RISC by cleaving their corresponding miRNA star strands. This phenomenon may depend on the secondary structure near the scissile phosphate of the miRNA duplex. In addition, I show that ATP is not required for star-strand cleavage in this process. Taken together, the data here provide insight into the miRNA-loading mechanisms in mammals.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.5975-5979
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• 2012

Background: To investigate the influence of telomerase activity, apoptosis, radiosensitivity of cervical cancer after siRNA-mediated knockdown of telomerase RNA and evaluate in vivo growth with gene interference. Methods: We studied siRNA-targeting-telomerase RNA transfection into the Hela cell line. Expression of hTERC mRNA was detected by RT-PCR and telomerase activity was measured by the TRAP assay. Growth inhibition was determined by MTT assay and radiosensitivity of the cervical cancer cells was examined by colony formation assay. In addtion, effects of hTERC inhibition in vivo were studied by injection of siRNA-transfected Hela cells into nude mice. Results: The hTERC siRNA effectively downregulated the expression of hTERC mRNA and also reduced the telomerase activity to 30% of the untreated control vlaue. The viability of hTERC siRNA transfected Hela cells was reduced by 44.7% after transfection. After radiation treatment, the radiosensitivity of Hela cells with hTERC knockdown was increased. In vivo, the tumors developing from the hTERC siRNA-transfected cells were of reduced size, indicating that the hTERT siRNA also depressed the tumorigenic potential of the Hela cells. Conclusions: Our results supported the concept of siRNA-mediated inhibition of telomerase mRNA which could inhibit the expression of hTERC and telomerase activity. Furthermore, radiosensitivity was upregulated after knockdown the hTERC in vivo and in vitro.

• Tuberculosis and Respiratory Diseases
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• v.56 no.4
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• pp.393-404
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• 2004

Background : Nucleic acid hybridization has become an essential technique in the development of our understanding of gene structure and function. The quantitative analysis of hybridization has been used in the measurement of genome complexity and gene copy number. The filter hybridization assay is rapid, sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. Methods : The authors assessed the accuracy, linearity, correlation coefficient and specificity of the hybridization depending on the added dose(0, 1, 5, and $10{\mu}g$) of non-specific rat spleen RNA to hybridization of surfactant protein A mRNA. Filter hybridization assays were used to obtain the equation of standard curve and thereby to quantitate the mRNA quantitation. Results : 1. Standard curve equation of filter hybridization assay between counts per minute (X) and spleen RNA input (Y) was Y=0.13X-19.35. Correlation coefficient was 0.98. 2. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) was Y=0.00066X-0.046. Correlation coefficient was 0.99. 3. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $1{\mu}g$ spleen RNA was Y=0.00056X-0.051. Correlation coefficient was 0.99. 4. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $5{\mu}g$ spleen RNA was Y=0.00065X-0.088. Correlation coefficient was 0.99. 5. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $10{\mu}g$ spleen RNA was Y=0.00051X-0.10. Correlation coefficient was 0.99. Conclusions : Comparison of cpm/filter in a linear range allowed accurate and reproducible estimation of surfactant protein A mRNA copy number irrespective of the addition dosage of non-specific rat spleen RNA over the range $0-10{\mu}g$.