• Animal cells and systems
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• v.8 no.3
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• pp.213-220
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• 2004

MicroRNA and siRNA (small interfering RNA), representative members of small RNA, exert their effects on target gene expression through association with protein complexes called miRNP (microRNA associated ribonucleoproteins) and RISC (RNA induced silencing complex), respectively. Although the protein complexes are yet to be fully characterized, human EIF2C2 protein has been identified as a component of both miRNP and RISC. In this report, we raised antiserum against EIF2C2 in order to begin understanding the protein complexes. An immunoblot result indicates that EIF2C2 protein is ubiquitously expressed in a variety of cell lines from human and mouse. EIF2C2 protein exists in both cellular compartments, as indicated by an immunoblot assay with a nuclear extract and a cytosolic fraction (S100 fraction) from HeLa S3 lysate. Depletion of EIF2C1 or EIF2C2 protein resulted in a decrease of microRNA, suggesting a possible role of these proteins in microRNA stability or biogenesis. We also prepared antiserum against dsRNA binding protein PACT, whose homologs in C. elegans and Drosophila are known to have a role in the RNAi (RNA interference) pathway. The expression of PACT protein was also observed in a wide range of cell lines.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1303-1307
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• 2007

A tumor suppressor, merlin is a member of ERM family proteins. It consists of N-terminal FERM domain, ${\alpha}-helical$ region, and C-terminal domain. Alternative splicing of merlin's mRNA generates two isotypes of merlin. Isotype I, which has exon17 at the C-terminus instead of exon16 in isotype II, is known to have tumor suppressor activity. Like other ERM proteins, the C-terminal domain of merlin isotype I interacts to its FERM domain. That of isotype II, however, was reported not to bind FERM domain despite the large common part of C-terminal domain, which possibly binds FERM domain. Here, we show the binding of FERM domain to both C-terminal domains of merlin's two isotypes by isothermal titration calorimetry. These results support that merlin isotype II also can form a closed conformation or a multimer by intramolecular or intermolecular interactions using their FERM domain and C-terminal domain.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8467-8471
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• 2016

Background: One of the major mechanisms for drug resistance is associated with altered anticancer drug transport, mediated by the human-adenosine triphosphate binding cassette (ABC) transporter superfamily proteins. The overexpression of adenosine triphosphate binding cassette, sub-family B, member 1 (ABCB1) by multidrug-resistant cancer cells is a serious impediment to chemotherapy. In our study we have studied the possibility that structural single-nucleotide polymorphisms (SNP) are the mechanism of ABCB1 overexpression. Materials and Methods: A total of 101 gastric cancer multidrug resistant cases and 100 controls were genotyped with sequence-specific primed PCR (SSP-PCR). Gene expression was evaluated for 70 multidrug resistant cases and 54 controls by real time PCR. The correlation between the two groups was based on secondary structures of RNA predicted by bioinformatics tool. Results: The results of genotyping showed that among 3 studied SNPs, rs28381943 and rs2032586 had significant differences between patient and control groups but there were no differences in the two groups for C3435T. The results of real time PCR showed over-expression of ABCB1 when we compared our data with each of the genotypes in average mode. Prediction of secondary structures in the existence of 2 related SNPs (rs28381943 and rs2032586) showed that the amount of ${\Delta}G$ for original mRNA is higher than the amount of ${\Delta}G$ for the two mentioned SNPs. Conclusions: We have observed that 2 of our studied SNPs (rs283821943 and rs2032586) may elevate the expression of ABCB1 gene, through increase in mRNA stability, while this was not the case for C3435T.

• BMB Reports
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• v.38 no.4
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• pp.432-439
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• 2005

$Ca^{2+}$/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of $Ca^{2+}$-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic $Ca^{2+}$-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stress induced expression of OsCam1 gene requires the $[Ca^{2+}]_{cyt}$ elevation that is known to occur in response to these stimuli.

• Molecules and Cells
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• v.21 no.1
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• pp.63-75
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• 2006

The 5′-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.3
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• pp.197-208
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• 2023

Dysregulation of certain long non-coding RNAs may facilitate tumor initiation and progression. However, numerous carcinogenesis-related long noncoding RNAs have not been characterized. The goal of this study was to elucidate the role of LINC00562 in gastric cancer (GC). The expression of LINC00562 was analyzed using real-time quantitative PCR and Western blotting. The proliferative capacity of GC cells was determined using Cell Counting Kit-8 and colony-formation assays. The migration of GC cells were evaluated using wound-healing assays. The apoptosis of GC cells was assessed by measuring the expression levels of apoptosis-related proteins (Bax and Bcl-2). Xenograft models in nude mice were constructed for in vivo functional analysis of LINC00562. The binding relationship between miR-4636 and LINC00562 or adaptor protein complex 1 sigma 3 (AP1S3), obtained from public databases, was confirmed using dual-luciferase and RNA-binding protein immunoprecipitation experiments. LINC00562 was expressed in GC cells at high levels. Knockdown of LINC00562 repressed GC cell growth and migration, promoted apoptosis in vitro, and inhibited tumor growth in nude mouse models. LINC00562 directly targeted miR-4636, and miR-4636 depletion restored the GC cell behavior inhibited by LINC00562 absence. AP1S3, an oncogene, binds to miR-4636. MiR-4636 downregulation increased AP1S3 level, restoring GC cell malignant behaviors inhibited by AP1S3 downregulation. Thus, LINC00562 exerts carcinogenic effects on GC development by targeting miR-4636-mediated AP1S3 signaling.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.76.1-76
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• 2003

The coat protein (CP) of Turnip crinkle virus (TCV) is organized into 3 distinct domains, R domain (RNA-binding) connected by an arm, 5 domain and P domain. We have previously shown that the CP of TCV strongly suppresses RNA silencing, and have mapped N-terminal R domain of which is also the elicitor of resistance response in the Arabidopsis ecotype Di-17 carrying the HRT resistance gene. In order to map the region in the TCV CP that is responsible for silencing suppression, a series of CP mutants were constructed, transformed into Agrobacterium, coinfiltrated either with HC-Pro (the helper component proteinase of tobacco etch potyvirus) known as a suppressor of PTGS or GFP constructs into leaves of Nicotiana benthmiana expressing GFP transgenically. In the presence of HC-Pro, all CP mutants were well protected, accumulating mutant CP mRNAs and their proteins even 5 days post-infiltration (DPI). In the presence of GFP, some mutant constructs which showed the accumulation of CP mutants and GFP mRNAs at early stage but eventually degraded at 5 DPI. Only a mutant which carrying 4 amino acid deletion of R domain was tolerable to maintain suppressing activity, suggesting that the suppressing activity is not directly related with the eliciting activity. A transient assay also revealed that the mutants synthesized their proteins, suggesting that a full length of CP sequences and its intact structure are required to stabilize CP, which suppresses the RNA silencing.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.195-202
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• 2011

Maintenance of adequate telomere length in developing cells is the most important concern to preserve the integrity of the genome. The length of telomere is strictly regulated by numerous telomere-binding proteins and/or interacting factors. Even though the expression of telomerase in the male reproductive tract has been characterized, developmental expressional profiling of telomerase and other telomere-associated proteins has not been determined in detail. The present study was attempted to examine expression patterns of catalytic subunit (Tert) and RNA component (Terc) of telomerase and two telomerase associated factors, telomerase associated protein 1 (Tep1) and TERF1 (TRF1) interacting nuclear factor 2 (Tinf2) in the testis and seminal vesicle of male rat during postnatal development. The real-time PCR analysis was utilized to quantify mRNA expression of molecules. The abundance of Tep1 mRNA in the testis and seminal vesicle was the highest at 5 months of age. Expressional fluctuation of Tinf2 during postnatal development was found in the testis, while expression of Tinf2 in the seminal vesicle was gradually increased until 5 months of age and then significantly decreased later. mRNA level of Tert gene in the testis was significantly increased at the adult and the elder, while the highest expression of Tert gene in the seminal vesicle was found at 5 months of age. Expression of Terc transcript in the testis and seminal vesicle was the highest at 5 months of age, followed by significant reduction at 1 and 2 years of ages. Such differential gene expression of telomere-associated factors and telomerase components in different male reproductive tissues during postnatal development indicates that maintenance of telomere length would be regulated in tissue- and/or age-specific manners.

• Journal of Korean Neurosurgical Society
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• v.29 no.2
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• pp.155-166
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• 2000

This study was designed to investigate the underlying mechanisms for the temporal changes of the striatal dopamine D2 receptors in the rat model of parkinsonism. After injection of the 6-hydroxydopamine into the substantia nigra of adult rats, we measured the receptor binding capacity(Bmax), mRNA and protein of the D2 receptor at 2, 4 and 8 weeks. Following the lesion, mRNA and protein were elevated simultaneously on both sides of the striata. They showed more increase on the normal side at 2 and 4 weeks, and then they were almost equally abundant on both sides at 8 weeks. We also observed their increased production in the diffuse cortical and subcortical regions. The Bmax value also increased bilaterally in both striata, and was higher on the normal side at 2 weeks and then on the lesioned side at 4 and 8 weeks. These findings suggest that production of the striatal D2 receptor is regulated at the transcriptional level in this animal model. They also imply that this control may be mediated through a pathway which can have influence on the whole brain, rather than the local control of the dopamine content alone. The measured functional activity(Bmax) of the D2 receptor was not proportional to the amount of the receptor mRNA and proteins produced. This difference may be explained by the post-translational modification of the receptor proteins, which may be controlled by such factor as the local concentration of dopamine.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.1012-1022
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• 2020

Objective: Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. Methods: MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. Results: In MAC-T cells, ATRA increased the mRNA levels of α_S1-casein and β-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 β (STAT5-β) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-β and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. Conclusion: ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.