• 대한의생명과학회지
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• 제29권4호
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• pp.231-241
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• 2023

Messenger RNA (mRNA)-based vaccines and treatments have recently emerged as a promising strategy. Naked mRNA presents various limitations for direct delivery. Therefore, in this paper, Lipid Nanoparticles (LNPs) were utilized for the delivery of mRNA. Lipid nanoparticle (LNP) mRNA systems are highly effective as vaccines, but their efficacy for pulmonary delivery has not yet been fully established. Additionally, research on effective delivery systems and administration methods for vaccines is required to resolve the stability and degradation issues associated with naked mRNA delivery. This study aimed to determine mRNA delivery efficiency via the inhalation of a lipid nanoparticle (LNP) formulation designed specifically for pulmonary delivery. To this purpose, we built a library of seven LNP configurations with different lipid molar and N/P ratios and evaluated their encapsulation efficiency using gel retardation assay. Among the tested LNPs, LNP1, LNP2-2, and LNP3-2 demonstrated high transfection efficiency in vitro based on FACS analyses luciferase assays, and intracellular accumulation tests. The mRNA delivery efficiencies of the selected LNPs after inhalation and intravenous injection were compared and evaluated. LNP2-2 showed the highest mRNA expression in healthy mouse lungs when aerosolized and was found to be non-toxic. These results indicate that LNP2-2 is a promising carrier for lung mRNA delivery via inhalation.

• BMB Reports
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• 제50권4호
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• pp.158-159
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• 2017

MicroRNAs (miRNAs) regulate gene expression by guiding the Argonaute (Ago)-containing RNA-induced silencing complex (RISC) to specific target mRNA molecules. It is well established that miRNAs are stabilized by Ago proteins, but the molecular features that trigger miRNA destabilization from Ago proteins remain largely unknown. To explore the molecular mechanisms of how targets affect the stability of miRNAs in human Ago (hAgo) proteins, we employed an in vitro system that consisted of a minimal hAgo2-RISC in HEK293T cell lysates. Surprisingly, we found that miRNAs are drastically destabilized by binding to seedless, non-canonical targets. We showed that miRNAs are destabilized at their 3' ends during this process, which is largely attributed to the conformational flexibility of the L1-PAZ domain. Based on these results, we propose that non-canonical targets may play an important regulatory role in controlling the stability of miRNAs, instead of being regulated by miRNAs.

• Tuberculosis and Respiratory Diseases
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• 제42권4호
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• pp.522-534
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• 1995

연구배경: 내독소에 의한 급성 폐손상의 발병기전에서 산소기가 중요한 역할을 한다는 사실은 잘 알려져 있다. 세포내에는 이러한 산소기에 의한 세포의 손상을 방지하는 정상 방어기전으로 여러 항산화효소가 존재하는데, 이중 SOD는 세포대사과정이나 외부 자극에 의해 생성된 superoxide로부터 세포의 손상을 방지하는 역할을 한다. 세포내 SOD는 주로 이중체의 구조로 세포질에 존재하는 CuZnSOD와 사중체의 구조로 미토콘드리아에 존재하는 MnSOD의 두 종류가 알려져 있으나, 폐포대식세포에서의 SOD mRNA 발현 및 그 조절기전에 대해서는 확실히 규명되어 있지 않다. 본 연구의 목적은 백서의 폐포대식세포에서 내독소 자극에 의한 MnSOD와 CuZnSOD mRNA 발현양상을 관찰하고 내독소 자극시 니타나는 SOD mRNA 발현의 조절기전을 규명하는데 있다. 방법: 백서의 기관지폐포세척액에서 얻은 세포를 plastic plate에 부착시켜 폐포대식세포를 분리한 후 내독소를 자극하여 내독소 용량($0.01{\mu}g/ml{\sim}10{\mu}g/ml$)과 자극시간(0, 2, 4, 8, 24 hrs)에 따른 MnSOD와 CuZnSOD MnSOD 발현양상을 Northern blot analysis를 시행하여 관찰하였다. 다음 단계로 MsSOD와 CuZnSOD mRNA 발현의 조절기전을 밝히고자 폐포대식세포를 각각 AD($5{\mu}g/ml$) 또는 CHX($5{\mu}g/ml$)로 전처치한 후 내독소로 자극하여 MnSOD와 CuZnSOD mRNA의 발현양상을 관찰하였다. 한편 내독소 투여가 SOD mRNA의 안정성을 변화시키는지 여부를 평가하기 위해 폐포대식세포를 대조군과 투여군으로 나누어 SOD mRNA의 분해속도를 비교하였다. 총 세포내 RNA는 guanidinium thiocyanate/phenol/chloroform법을 이용하여 추출하였고, Northern blot analysis는 $^{32}P$로 표지된 백서의 MnSOD와 CuZnSOD cDNAs를 이용하여 시행하였다. 결과: 백서의 폐포대식세포에서 MnSOD mRNA의 발현은 내독소 투여량의 증가세 따라 증가되었고 내독소를 투여하고 8시간후에 정점을 이루었으나, CuZnSOD mRNA의 발현은 내독소의 용량 및 투여후 반응시간에 따라 변화하지 않았다. 내독소 투여후 MnSOD mRNA의 발현증가는 AD 또는 CHX 각각의 전처치에 의해 모두 억제되었다. MnSOD mRNA의 안정성은 내독소 투여에 의해 변화하지 않았다. 결론: 이상의 결과로 백서의 폐포대식세포는 내독소 자극에 반응하여 SOD를 생성하는 중요세포이고, 내독소에 의한 MnSOD mRNA의 발현은 전사단계에서 조정되며 mRNA의 안정성을 변화시키지 않고 새로운 단백의 합성이 필요한 것으로 사료된다.

• BMB Reports
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• 제47권11호
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• pp.619-624
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• 2014

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

• Molecules and Cells
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• 제42권5호
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• pp.426-439
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• 2019

Many small RNAs (sRNAs) regulate gene expression by base pairing to their target messenger RNAs (mRNAs) with the help of Hfq in Escherichia coli. The sRNA DsrA activates translation of the rpoS mRNA in an Hfq-dependent manner, but this activation ability was found to partially bypass Hfq when DsrA is overproduced. The precise mechanism by which DsrA bypasses Hfq is unknown. In this study, we constructed strains lacking all three rpoS-activating sRNAs (i.e., ArcZ, DsrA, and RprA) in $hfq^+$ and $Hfq^-$ backgrounds, and then artificially regulated the cellular DsrA concentration in these strains by controlling its ectopic expression. We then examined how the expression level of rpoS was altered by a change in the concentration of DsrA. We found that the translation and stability of the rpoS mRNA are both enhanced by physiological concentrations of DsrA regardless of Hfq, but that depletion of Hfq causes a rapid degradation of DsrA and thereby decreases rpoS mRNA stability. These results suggest that the observed Hfq dependency of DsrA-mediated rpoS activation mainly results from the destabilization of DsrA in the absence of Hfq, and that DsrA itself contributes to the translational activation and stability of the rpoS mRNA in an Hfq-independent manner.

• Molecules and Cells
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• 제46권1호
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• pp.48-56
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• 2023

Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in post-transcriptional gene expression, regulating mRNA stability, translation, and localization. Similar to DNA mutations producing aberrant proteins, RNA alterations expand the transcriptome landscape and change the cellular proteome. Recent global analyses reveal that many genes express various forms of altered RNAs, including 3'UTR length variants. Alternative polyadenylation and alternative splicing are involved in diversifying 3'UTRs, which could act as a hidden layer of eukaryotic gene expression control. In this review, we summarize the functions and regulations of 3'UTRs and elaborate on the generation and functional consequences of 3'UTR diversity. Given that dynamic 3'UTR length control contributes to phenotypic complexity, dysregulated 3'UTR diversity might be relevant to disease development, including cancers. Thus, 3'UTR diversity in cancer could open exciting new research areas and provide avenues for novel cancer theragnostics.

• 미생물학회지
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• 제47권4호
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• pp.289-294
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• 2011

Bacillus subtilis에 존재하는 DEAD-box RNA helicase 유전자의 결손이 저온 충격에 민감성을 보이는지를 조사하였다. 저온 충격에 민감한지를 알아 보기 위하여 대수기에($O.D_{600}$=0.5-0.6) 있는 세포를 $15^{\circ}C$도 낮추어 저온충격을 가하여 생장하는 정도를 조사하였다. DEAD-box RNA helicase 유전자 ydbR, yfmL, yqfR, deaD의 결손 균주들이 저온충격을 가하였을 때 ydbR 결손 균주의 생장이 야생형 균주에 비해 5배 정도 현저히 감소하였으나, 다른 DEAD-box RNA helicase 유전자의 (yfmL, yqfR, deaD) 결손은 야생형 균주와 비슷한 생장을 보였다. 저온에서의 유전자 발현을 알아보기 위하여 Northern blot으로 mRNA 양을 알아본 결과 $37^{\circ}C$에 비해 $15^{\circ}C$에서 ydbR과 yqfR의 mRNA전사물 증가를 확인할 수 있었고, 반면에 yfmL과 deaD의 전사 증가는 관찰되지 않았다. $37^{\circ}C$에서 $15^{\circ}C$로 저온 충격을 가하면 ydbR mRNA 양의 뚜렷한 증가를 확인하였고, 전사 억제제인 rifampicin를 처리하여 ydbR mRNA의 양을 조사하였을 때 $15^{\circ}C$ 조건에서는 mRNA 양이 거의 유지하는 반면에 $37^{\circ}C$ 조건에서는 급격한 mRNA의 감소가 일어나 전사과정에서 유도되기 보다는 전사 후 전사물의 안정에 기인하는 것으로 보인다. 이와 관련하여 ydbR 유전자의 5' UTR (untranaslated region) 부근에서 csp (cold shock protein) 유전자에서 관찰되는 cold box element를 확인하였고, ydbR이 저온 충격 조건에서 발현되는 과정이 csp와 유사하게 전사물의 안정성에 기인함을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.599-605
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• 2000

Lipopolysaccharide (LPS) is responsible for the tissue injury that occurs following the invasion of multicelluar organisms by Gram-negative microbes. The effect of LPS on IFN-$\gamma$-induced chemokine Mig gene expression in mouse peritoneal macrophages was investigated. Very little Mig mRNA was detectable upon exposure to LPS without IFN-$\gamma$. Although LPS alone is only minimally effective, LPS plus IFN-$\gamma$ synergized to produce a high level of Mig mRNA in the peritoneal macrophages. This synergy was not dependent on a new protein synthesis, and was not controlled at the level of the gene transcription. Futhermore, LPS did not increase IFN-$\gamma$-induced Mig mRNA stability. Accordingly, it is suggested the LPS may synergize the expression of IFN-$\gamma$-induced Mig mRNA through a process that depends on a pretranscriptional level or concurrent Mig mRNA translation.

• Genomics & Informatics
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• 제12권2호
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• pp.71-75
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• 2014

The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA $m^1G37$ methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, $m^1G37$ modification was reported to take place on three conserved tRNA subsets ($tRNA^{Arg}$, $tRNA^{Leu}$, $tRNA^{Pro}$); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the $m^1G37$ modification. The present study reveals Gm18, $m^1G37$ modification, and positions of $m^1G$ that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the $m^1G$ and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs ($tRNA^{Met}$, $tRNA^{Pro}$, $tRNA^{Val}$). Whereas the $m^1G37$ modification base G is formed only on $tRNA^{Arg}$, $tRNA^{Leu}$, $tRNA^{Pro}$, and $tRNA^{His}$, the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and $m^1G$ modification occur irrespective of a G residue in tRNAs.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.882-889
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• 2021

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.