• Title/Summary/Keyword: hnRNP K

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Cloning of hnRNP E1 cDNA via yeast two-hybrid system and a study on protein-protein interaction between hnRNP E1 and hnRNP K (이스트 two-hybrid 시스템을 이용한 hnRNP E1 cDNA의 클로닝과 hnRNP E1-hnRNP K 상호결합에 대한 연구)

  • Choi, Mie-Young
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.9 no.6
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    • pp.1795-1799
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    • 2008
  • The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a component of hnRNP complexes. This protein binds strongly to cytidine-rich RNA/DNA sequences. It is a nucleocytoplasmic shuttling protein. To investigate the functions of hnRNP K, I searched for hnRNP K-interacting proteins in HeLa cDNA library using a yeast two-hybrid screening system. One of the cDNA clones is identical to human hnRNP E1 (poly(rC) binding protein 1) cDNA (GenBank accession number XM_031585). In this study, hnRNP K is shown to specifically interact with hnRNP E1 in yeast two-hybrid system and in vitro biochemical assay.

Analysis of the Role of RGG box of human hnRNP A1 protein (인간 hnRNP A1 단백질에 포함된 RGG 상자의 기능 분석)

  • Choi, Mieyoung
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.18 no.12
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    • pp.575-580
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    • 2017
  • This study analyzed the effects of RGG box of hnRNP A1 on its subcellular localization and stabilization of hnRNP A1 over a three year period from October 2014. First, a 6R/K mutation in RGG box was generated, and pcDNA1-HA-hnRNP A1(6R/K) was constructed. The subcellular localization of hnRNP A1(6R/K) from the HeLa cells transfected with this plasmid DNA was analyzed by immunofluorescence microscopy. HA-hnRNP A1(6R/K) was found to exhibit nuclear and cytoplasmic fluorescence. The stability of hnRNP A1(6R/K) was checked by Western blot analysis using the expressed protein from the HeLa cells transfected with the pcDNA1-HA-hnRNP A1(6R/K). The results show that HA-hnRNP A1(6R/K) has a smaller size. These confirm that HA-hnRNP A1(6R/K) is localized both in the nuclear and cytoplasm, not because 6R/K mutation affects the nuclear localization of hnRNP A1, but because 6R/K mutation causes hnRNP A1(6R/K) to cleave at the mutation or near the mutation site. The cleaved protein fragment, which lacks the M9 domain (i.e. nuclear localization signal of hnRNP A1), did not exhibit nuclear fluorescence. This suggests that the arginines of RGG box in hnRNP A1 play an important role in stabilizing hnRNP A1. An analysis of the RNA-binding ability of hnRNP A1(6R/K) expressed and purified from bacteria will be a subsequent research project.

Identification of the Interaction between Insulin-like Growth Factor Binding Protein-4 (IGFBP-4) and Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) (IGF결합 단백질-4(IGFBP-4)와 이질 핵 리보핵산단백질 L (hnRNP L)의 상호결합의 식별)

  • Choi, Mieyoung
    • Journal of Life Science
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    • v.23 no.11
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    • pp.1311-1316
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    • 2013
  • Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a major pre-mRNA binding protein and it is an abundant nuclear protein that shuttles between the nucleus and the cytoplasm. hnRNP L is known to be related to many cellular processes, including chromatin modification, pre-mRNA splicing, mRNA export of intronless genes, internal ribosomal entry site (IRES)-mediated translation, mRNA stability, and spermatogenesis. In order to identify the cellular proteins interacting with hnRNP L, this study performed a yeast two-hybrid screening, using a human liver cDNA library. The study identified insulin-like growth factor binding protein-4 (IGFBP-4) as a novel interaction partner of hnRNP L in the human liver. It then discovered, for the first time, that hnRNP L interacts specifically with IGFBP-4 in a yeast two-hybrid system. The authenticity of this two-hybrid interaction of hnRNP L and IGFBP-4 was confirmed by an in vitro pull-down assay.

Effect of Modulation of hnRNP L Levels on the Decay of bcl-2 mRNA in MCF-7 Cells

  • Lim, Mi-Hyun;Lee, Dong-Hyoung;Jung, Seung-Eun;Youn, Dong-Ye;Park, Chan-Sun;Lee, Jeong-Hwa
    • 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.

Human Ribosomal Protein L18a Interacts with hnRNP E1

  • Han, Sun-Young;Choi, Mie-Young
    • Animal cells and systems
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    • v.12 no.3
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    • pp.143-148
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    • 2008
  • Heterogeneous nuclear ribonucleoprotein E1(hnRNP E1) is one of the primary pre-mRNA binding proteins in human cells. It consists of 356 amino acid residues and harbors three hnRNP K homology(KH) domains that mediate RNA-binding. The hnRNP E1 protein was shown to play important roles in mRNA stabilization and translational control. In order to enhance our understanding of the cellular functions of hnRNP E1, we searched for interacting proteins through a yeast two-hybrid screening while using HeLa cDNA library as target. One of the cDNA clones was found to be human ribosomal protein L18a cDNA(GenBank accession number BC071920). We demonstrated in this study that human ribosomal protein L18a, a constituent of ribosomal protein large subunit, interacts specifically with hnRNP E1 in the yeast two-hybrid system. Such an interaction was observed for the first time in this study, and was also verified by biochemical assay.

Tra2${\alpha}$ and hnRNP K might be Functional Partners of Rbm for Regulation of RNA Processes during Spermatogenesis

  • Lee, Jungmin;Kim, Euisu;Jang, Sung Key;Rhee, Kunsoo
    • Animal cells and systems
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    • v.8 no.1
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    • pp.65-70
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    • 2004
  • Rbm is a male infertility gene located in the AZFb region of the Y chromosome. Expression pattern of Rbm indicates that Rbm is critical for early phase of male germ cell development. It shares strong structural homology with hnRNP G, suggesting a function as an RNA processing factor. In order to gain a clue on the molecular mechanisms of Rbm on male germ cell development, we examined interactions of Rbm with selected proteins in yeast. The results revealed specific interactions between Rbm, hnRNP K and Tra2${\alpha}$. These results suggest that hnRNP K and Tra2${\alpha}$ may be functional partners of Rbm in male germ cells. We propose a model in which hnRNP K may playa role as a platform for Rbm and Tra2${\alpha}$.

The Schizosaccharomyces pombe Proteins that Bind to the Human HnRNPA1 Winner RNA

  • Kim, Jeong-Kook
    • Journal of Microbiology
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    • v.35 no.4
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    • pp.327-333
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    • 1997
  • Although extensively characterized in human cells, no heterogeneous nuclear ribonucleoprotein(hnRNP) has been found in the fission yeast Schizosaccharomyces pombe which is amenable to genetic studies and more similar to mammals than Saccharomyces cerevisiae is in terms of RNA processing. As a first step to characterize hnRNPs from S. pombe, attempt was made to find human hnRNP A1 homologs from S. pombe. The RNA molecule (A1 winner) containing the consensus high-affinity hnRNP A1 binding site (UAGGGA/U) was synthesized in vitro and used in an ultraviolet(UV) light-induced protein-RNA cross-linking assay. A number of S, pombe proteins bound to the A1 winner RNA. An approximately 50-kDa protein(p50) cross-linked more efficiently to the A1 winner RNA than other proteins. The p50 protein did not cross-link to a nonspecific RNA, but rather to the A1-5’ SS RNA in which the consensus 5’ splice junction sites of S. pombe introns were abolished. This suggests that the p50 protein, however, did not bind to the single-stranded DNA to shich the human hnRNP A1 could bind and be eluted with 0.5M NaCl. Further analysis should reveal more features of this RNA-binding protein.

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Identifying the cellular location of brain cytoplasmic 200 RNA using an RNA-recognizing antibody

  • Shin, Heegwon;Lee, Jungmin;Kim, Youngmi;Jang, Seonghui;Ohn, Takbum;Lee, Younghoon
    • BMB Reports
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    • v.50 no.6
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    • pp.318-322
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    • 2017
  • Brain cytoplasmic 200 RNA (BC200 RNA) is a neuron-specific non-coding RNA, implicated in the inhibition of local synaptodendritic protein synthesis, and is highly expressed in some cancer cells. Although BC200 RNA has been shown to inhibit translation in vitro, the cellular location of this inhibition is unknown. In this study, we used a BC200 RNA-recognizing antibody to identify the cellular locations of BC200 RNA in HeLa cervical carcinoma cells. We observed punctate signals in both the cytoplasm and nucleus, and further discovered that BC200 RNA co-localized with the p-body decapping enzyme, DCP1A, and the heterogeneous nuclear ribonucleoprotein E2 (hnRNP E2). The latter is a known BC200 RNA-binding partner protein and a constituent of p-bodies. This suggests that BC200 RNA is localized to p-bodies via hnRNP E2.

Effect of hnRNP-like protein THO4 on growth and mRNA export in fission yeast (분열효모에서 hnRNP-유사 단백질인 THO4가 생장 및 mRNA 방출에 미치는 영향)

  • Park, Jin Hee;Lee, Sojeong;Yoon, Jin Ho
    • Korean Journal of Microbiology
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    • v.54 no.2
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    • pp.91-97
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    • 2018
  • The evolutionally conserved TREX complex member, Yra1/ALY, belongs to the REF (RNA and export factor binding proteins) family of hnRNP-like proteins, which has been implicated in multiple processes including transcription, nuclear RNA stability, and mRNA export. Fission yeast, Schizosaccharomyces pombe, genome encodes two members of REF proteins. In addition to Mlo3 known previously as an mRNA export factor, there is the other REF protein, Tho4, which is predicted as a component of THO complex. Here we showed that deletion of tho4 (SPBC106.12c) gene does not inhibit both growth and nuclear mRNA export. However, overexpression of tho4 displays growth retardation and slight accumulation of $poly(A)^+$ RNA in the nucleus. Neither ${\Delta}tho4$ ${\Delta}mlo3$ nor ${\Delta}tho4$ ${\Delta}mex67$ double mutants exhibit additive growth defect. Moreover, yeast two-hybrid and co-immunoprecipitation analysis did not show that the Tho4 protein interacted with any members of TREX complex and mRNA export factor Rae1. Contrary to expectation, these observations support that the S. pombe Tho4 is not a component of TREX complex, and not directly involved in bulk mRNA export from the nucleus.