• Journal of Life Science
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• v.24 no.10
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• pp.1144-1150
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• 2014

The eIF4E protein is the key regulator of translation initiation. The interaction of eIF4E with eIF4G triggers the translation of mRNA, and several proteins interrupt this association to modulate translation. Human 4E-T is one of the eIF4E-binding partners that represses the translation of bound mRNAs, and it is involved in the transport of eIF4E to processing bodies (P-bodies). Although Clast4, the mouse homolog of human 4E-T, might play critical roles in the regulation of translation, its properties are not well known. In this report, we deciphered the properties of Clast4 by determining its phosphorylation state, binding to eIF4E, and effects of overexpression on cell proliferation. Clast4 was phosphorylated by protein kinase A (PKA) in vivo on several residues of its amino terminus. Nevertheless, the PKA phosphorylation of Clast4 appeared to have no effect on either its eIF4E-binding ability or localization. Clast4 interacted with eIF4E1 and CPEB. The conserved eIF4E-binding sequence in Clast4, $YXXXXL_{\phi}$, was important for binding eIF4E1A but not eIF4E1B. Similar to that of another well-known eIF4E regulator, the eIF4E binding protein (4E-BP), the overexpression of Clast4 decreased cell proliferation. These results suggest that Clast4 acts as a global translation regulator in cells.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.1
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• pp.133-140
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• 2005

Although amino acids are substrates for the synthesis of proteins and nitrogen-containing compounds, it has become more and more clear over the years that these nutrients are also extremely important as regulators of body protein turnover. The branched-chain amino acids (BCAAs) together or simply leucine alone stimulate protein synthesis and inhibit protein breakdown in skeletal muscle. However, it was only recently that the mechanism(s) involved in the regulation of protein turnover by BCAAs has begun to be defined. The acceleration of protein synthesis by these amino acids seems to occur at the level of peptide chain initiation. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Despite our knowledge of the induction of protein synthesis by BCAAs, there are few studies on the suppression of protein degradation. The recent findings that oral administration of leucine rapidly reduced $N^{\tau}$-methylhistidine (3-methylhistidine; MeHis) release from isolated muscle, an index of myofibrillar protein degradation, indicate that leucine suppresses myofiblilar protein degradation. The details of the molecular mechanism by which leucine inhibits proteolysis is just beginning to be elucidated. The purpose of this report was to review the current understanding of how BCAAs act as regulators of protein turnover.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.401-404
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• 2010

Tho1 is a RNA-binding protein that assembles co-transcriptionally onto the nascent mRNA and is thought to be involved in mRNP biogenesis and mature mRNA export to cytoplasm in budding yeast. In fission yeast Schizosaccharomyces pombe, a homologue of THO1 (spTho1) was identified based on sequence alignment. A deletion mutant in a diploid strain was constructed by replacing one of spTho1-coding region with an ura4+ gene using one-step gene disruption method. Tetrad analysis showed that the spTho1 was not essential for growth. The spTho1 mutant did not show any defects of bulk mRNA export. However, over-expression of spTho1 from strong nmt1 promoter caused the growth defects and accumulation of poly(A)$^+$ RNA in the nucleus. These results suggest that spTho1 is involved in mRNA export from the nucleus to cytoplasm though it is not essential.

• Development and Reproduction
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• v.14 no.4
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• pp.287-295
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• 2010

It was recently reported that nesfatin-1/NUCB2, which is secreted from the brain, controls appetite and energy metabolism. The purpose of this research was to confirm whether or not the protein and its binding site should have been expressed in the mouse reproductive organs and to know the possible effects of nesfatin-1 on the reproductive function. Using the ICR female mouse ovary and uterus, the expression of NUCB2 mRNA was confirmed with the conventional PCR and the relative amount of NUCB2 mRNA in the tissues was analyzed with real-time PCR. Immunohistochemical staining was performed using the nesfatin-1 antibody to investigate the nesfatin-1 protein expression and the biotin conjugated nesfatin-1 to confirm the binding site for nesfatin-1 in the ovary. Furthermore, in order to examine if the expression of NUCB2 mRNA in the ovary and uterus is affected by gonadotropin, its mRNA expression was analyzed after PMSG administration into mice. As a result, the expression level of NUCB2 mRNA in the ovary and the uterus was as much as the expression level in hypothalamus. As a result of the immunohistochemical staining, nesfatin-1 proteins were localized at the theca cells, the interstitial cells, and some of the luteal cells. However, the granulosa cells in the follicles did not stain. Interestingly, the oocytes in the some follicles were stained with nesfatin-1. On the other hand, nesfatin-1 protein binding sites were displayed at the theca cells and the interstitial cells near the tunica albuginea. After PMSG administration the expression level of NUCB2 mRNA was increased in the ovary and the uterus. These results demonstrate that for the first time the nesfatin-1 and its binding site were expressed in the ovary and NUCB2 mRNA expression was controlled by gonadotropin, suggesting an important role in the reproductive organs as a local regulator. Therefore, further study is needed to elucidate the functions of nesfatin-1 on the reproductive organs.

• Interdisciplinary Bio Central
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• v.2 no.3
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• pp.8.1-8.5
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• 2010

Introduction: Recently identified human homologues of ALKB protein have shown the activity of DNA damaging drugs, used for cancer therapy. Bioinformatics study of hABH2 and hABH3 had led to the discovery of a novel DNA repair mechanism. Very little is known about structure and function of hABH4, one of the members of this superfamily. Therefore, in present study we are intended to predict its structure and function through various bioinformatics tools. Materials and Methods: Modeling was done with modeler 9v7 to predict the 3D structure of the hABH4 protein. This model was validated with the program Procheck using Ramachandran plot statistics and was submitted to PMDB with ID PM0076284. The 3d2GO server was used to predict the functions. Residues at protein ligand and protein RNA binding sites were predicted with 3dLigandSite and KYG programs respectively. Results and Discussion: 3-D model of hABH4, ALKBH4.B99990003.pdb was predicted and evaluated. Validation result showed that 96.4 % residues lies in favored and additional allowed region of Ramachandran plot. Ligand binding residues prediction showed four Ligand clusters, having 24 ligands in cluster 1. Importantly, conserved pattern of Glu196-X-Pro198- Xn-His254 in the functional domain was detected. DNA and RNA binding sites were also predicted in the model. Conclusion and Prospects: The predicted and validated model of human homologue hABH4 resulted from this study may unveil the mechanism of DNA damage repair in human and accelerate the research on designing of appropriate inhibitors aiding in chemotherapy and cancer related diseases.

• Bulletin of the Korean Chemical Society
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• v.28 no.3
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• pp.427-431
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• 2007

In order to gain insight into the molecular changes at the protein level in plants exposed to low temperature for a long period of time (vernalization), proteome analyses of vernalization-treated Arabidopsis thaliana have been carried out by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Fourteen proteins including ATP binding/GTP binding/translation elongation factor and glycine-rich RNA-binding protein 7 (GRP7) showed differential expression in vernalization-treated Arabidopsis thaliana. GRP7 showed the most dramatic increase in expression suggesting its involvement in response to vernalization treatment.

• Proceedings of the Korea Information Processing Society Conference
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• 2016.10a
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• pp.583-586
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• 2016

단백질과 RNA의 상호작용 데이터가 대량으로 늘어남에 따라, 단백질과 RNA의 결합부위를 예측하는 계산학적인 방법들이 많이 개발되고 있다. 하지만, 많은 계산학적인 방법들은 단백질에서 단백질과 RNA 결합부위를 예측한다는 한계점이 있었다. 본 논문에서는 RNA와 단백질의 서열정보를 모두 사용하여, 단백질과 결합하는 RNA 결합부위를 예측하는 기법과 그 결과를 논한다. WEKA random forest(http://www.cs.waikato.ac.nz/ml/weka/)를 이용하여 예측 모델을 개발하였고, RNA 서열의 서열 프로파일, 서열 composition, 결합 상대방의 단백질의 특성 등을 특정으로 표현하였다. Random forest 기법을 사용한 cross validation의 결과로서 1:1 모델에서 제일 높은 성능인 92.4% sensitivity, 92.0% specificity, 92.2% accuracy를 보였고, independent test에서는 72.5% sensitivity, 90.0% specificity, 2.1% accuracy를 보였다.

• Journal of Microbiology
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• v.41 no.3
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• pp.239-247
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• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.414-421
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• 2014

This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of ${\alpha}s1$-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 ${\mu}M$) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 ${\mu}M$ in a concentration-dependent manner, and the addition of 600 ${\mu}M$ was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

• BMB Reports
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• v.43 no.5
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• pp.344-348
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• 2010

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Staufen2 is an mRNA-binding protein expressed in the cell bodies and cellular processes of different brain cells. It is notably involved in the transport of dendritic mRNAs along microtubules. Its knockdown expression was shown to change spine morphology and impair synaptic functions. However, the identity of Staufen2-bound mRNAs in brain cells is still completely unknown. As a mean to identify these mRNAs, we immunoprecipitated Staufen2-containing mRNPs from embryonic rat brains and used a genome wide approach to identify Staufen2-associated mRNAs. The genome wide approach identified 1780 mRNAs in Staufen2-containing mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. These results represent an additional and important step in the characterization of Staufen2- mediated neuronal functions in rat brains.