• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.227-231
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• 1998

The teleosts represent ancient real-bony vertebrates in phylogeny and resemble major genetic patterns to higher vertebrates. In the present study, we have defined the single family of insulin-like growth factors (IGFs) from flounder (Paralichthys olivaceus), compared to the prototype of IGFs observed in the Agnathan hagfish. In flounder, IGFs are clearly diverged into two major types including type I and II, and they are structurally similar by displaying a multidomain structure consisting of five functional regions as previously found in other vertebrates. However, flIGF-I appears to be more basic (pI 8.03) than the flIGF-II (pI 5.34) in the fully processed form for the B to D domain region. The flIGF-I seems to contain an evolutionary conserved Asn-linked glycosylation in E domain, which is not found in flIGF­II. The most interesting feature is that flIGF-II appeared to be structurally close to hagfish IGF in secondary structures, particularly in Band D domains. This could tell us an idea on the molecular divergence of IGFs from the Agnatha to teleosts during the vertebrate phylogeny. It also support, in part, a notion regarding on how IGF-II is appeared as more embryonic during development. Nonetheless, the biologically active B to D domain region of flIGF-II shows significant sequence homology of $65.6\%$ to flIGF-Is and contains the evolutionary conserved insulin-family signature, as well as a reserved recognition site (Lys) in D domain, necessary to generate proteolytic cleavage for E-peptide. A significant structural difference was found in E domain in which flIGF-I possesses two potential alternative splicing donor site at $Val^{17,\;24}$ of E domain. Therefore, it seems so far that IGF-I sorely produces spliced variants due to the spliced E-peptide moiety while IGF-II appears to be maintained in a single type during evolution. IGF-II, however, may be also possible to transcribe unidentified variants, depending on the physiological conditions of tissues in vertebrates in vivo.

• Molecules and Cells
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• v.21 no.1
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• pp.21-28
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• 2006

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase PKR plays a key role in interferon-mediated host defense against viral infection, and is implicated in cellular transformation and apoptosis. We have isolated a cDNA of simian PKR encoding a product with 83% amino acid identity to the human homolog and showed that PKR expression is significantly attenuated in the Vero E6 African green monkey kidney cells devoid of type I interferon genes. A variant form of PKR lacking the exon 12 in the kinase domain is produced in these cells, presumably from an alternatively spliced transcript. Unlike wild type PKR, the variant protein named PKR-${\Delta}E12$ is incapable of auto-phosphorylation and phosphorylation of eIF2-${\alpha}$, indicating that the kinase sub-domains III and IV embedded in exon 12 are indispensable for catalytic function. PKR-${\Delta}E12$ had no dominant negative effect but was weakly phosphorylated in trans by wild type PKR.

• Journal of Integrative Natural Science
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• v.6 no.4
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• pp.234-243
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• 2013

N-terminal kinase-like (NTKL) protein was initially identified as a protein binding to protein kinase B (PKB, also known as Akt). Though NTKL-BP1 (NTKL-binding protein 1) has been identified as an NTKL binding protein, its functions related to binding have not yet been elucidated. Here, a new alternative spliced variant of NTKL and its association with integrin ${\beta}1$ is described, in addition to the kinase activity of NTKL and its substrate candidates. Although the phosphorylation of the candidates must be further confirmed using other experimental methods, the observation that NTKL can phosphorylate ROCK1, DYRK3, and MST1 indicates that NTKL may act as a signaling protein to regulate actin assembly, cell migration, cell growth, and to facilitate differentiation and development in an integrin-associated manner.

• Molecules and Cells
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• v.26 no.2
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• pp.175-180
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• 2008

$I{\kappa}B$ kinase (IKK), the pivotal kinase in signal-dependent activation of nuclear factor-${\kappa}B$ (NF-${\kappa}B$), is composed of multiple protein components, including IKK ${\alpha}/{\beta}/{\gamma}$ core subunits. To investigate the regulation of the IKK complex, we immunoaffinity purified the IKK complex, and by MALDI-TOF mass spectrometry identified a splice variant of zinc finger protein 268 (ZNF268) as a novel IKKinteracting protein. Both the full-length and the spliced form of the ZNF268 protein were detected in a variety of mammalian tissues and cell lines. The genes were cloned and expressed by in vitro transcription/translation. Several deletion derivatives, such as KRAB domain (KRAB) on its own, the KRAB/spacer/4-zinc fingers (zF4), and the spacer/4-zinc fingers (zS4), were ectopically expressed in mammalian cells and exhibited had different subcellular locations. The KRAB-containing mutants were restricted to the nucleus, while zS4 was localized in the cytosol. TNF-${\alpha}$-induced NF-${\kappa}B$ activation was examined using these mutants and only zS4 was found to stimulate activation. Collectively, the results indicate that a spliced form of ZNF268 lacking the KRAB domain is located in the cytosol, where it seems to play a role in TNF-${\alpha}$-induced NF-${\kappa}B$ activation by interacting with the IKK complex.

• BMB Reports
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• v.40 no.5
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• pp.749-756
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• 2007

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5$\alpha$, Tssk5$\beta$, Tssk5$\gamma$ and Tssk5$\delta$. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5$\alpha$ which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5$\alpha$, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5$\alpha$ exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5$\beta$, TSSK5$\gamma$ and TSSK5$\delta$ cannot be stimulated at the CREB/CRE responsive pathway in comparison to TSSK5$\alpha$. These findings suggest that TSSK5$\beta$, TSSK5$\gamma$, TSSK5$\delta$ may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5$\beta$, TSSK5 $\gamma$ and TSSK5$\delta$ can directly interact with TSSK5$\alpha$. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1581-1590
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• 2018

Objective: The aim of this study was to clone alternative splicing isoforms of pig myoneurin (MYNN), predict the structure and function of coding protein, and study temporal and spatial expression characteristics of each transcript. Methods: Alternative splice isoforms of MYNN were identified using RNA sequencing (RNA-seq) and cloning techniques. Quantitative real-time polymerase chain reaction (qPCR) was employed to detect expression patterns in 11 tissues of Large White (LW) and Mashen (MS) pigs, and to study developmental expression patterns in cerebellum (CE), stomach (ST), and longissimus dorsi (LD). Results: The results showed that MYNN had two alternatively spliced isoforms, MYNN-1 (GenBank accession number: KY470829) and MYNN-2 (GenBank accession number: KY670835). MYNN-1 coding sequence (CDS) is composed of 1,830 bp encoding 609 AA, whereas MYNN-2 CDS is composed of 1,746 bp encoding 581 AA. MYNN-2 was 84 bp less than MYNN-1 and lacked the sixth exon. MYNN-2 was found to have one $C_2H_2$ type zinc finger protein domain less than MYNN-1. Two variants were ubiquitously expressed in all pig tissues, and there were significant differences in expression of different tissues (p<0.05; p<0.01). The expression of MYNN-1 was significantly higher than that of MYNN-2 in almost tissues (p<0.05; p<0.01), which testified that MYNN-1 is the main variant. The expression of two isoforms decreased gradually with increase of age in ST and CE of MS pig, whereas increased gradually in LW pig. In LD, the expression of two isoforms increased first and then decreased with increase of age in MS pig, and decreased gradually in LW pig. Conclusion: Two transcripts of pig MYNN were successfully cloned and MYNN-1 was main variant. MYNN was highly expressed in ST, CE, and LD, and their expression was regular. We speculated that MYNN plays important roles in digestion/absorption and skeletal muscle growth, whereas the specific mechanisms require further elucidation.

• Molecules and Cells
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• v.41 no.8
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• pp.733-741
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• 2018

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2153-2158
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• 2014

Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

• Development and Reproduction
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• v.5 no.1
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• pp.81-88
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• 2001

Gonadotropin-releasing hormone (GnRH) has been known to play a role in the regulation of hCG secretion by human placenta. Recently, a gene encoding the second f개m of GnRH (GnRH-II) was identified in human. Herein, we demonstrate that GnRH-II is expressed in human placenta and assess GnRH-II expression by nested RT-PCR and immunohistochemistry in human placenta during the first trimester. We found that two altematively spliced transcripts of GnW-II mRNA were expressed in human placental tissues of first trimester and the shorter variant had a 21-bp deletion in GnRH-associated peptide (GAP). Immunoreactive GnRH-II was localized in both cytotrophoblastic and syncytiotrophoblastic cytoplasm. The immunostaining intensity was stronger in cytotrophoblast. Villous stromal cells also showed GnRH-II immunoreactiyiry. The results of our study report that the second isoform of GnRH (GnRH-II) is expressed in the first trimester human placenta and we suggest that GnRH-II may also play a regulatory role in maintenance of early pregnancy and hCG secretion in human placenta.