hnRNPA1基因在家蚕翅原基组织中的表达与定位分析
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摘要
核不均一蛋白A1(hnRNPA1)是一个重要的RNA结合蛋白。本研究旨在获得家蚕hnRNPA1基因的cDNA,并对其在家蚕翅原基组织进行表达和定位分析。以家蚕幼虫期翅原基mRNA为模板通过反转录克隆家蚕BmhnRNPA1基因的全长cDNA,并对其进行生物信息学分析。构建pET32a-BmhnRNPA1蛋白表达载体,表达且纯化得到BmhnRNPA1重组蛋白,并制备该蛋白多克隆抗体,通过实时荧光定量RT-PCR、Western blot和免疫组化方法检测BmhnRNPA1在家蚕幼虫和蛹翅原基组织中的表达与定位。克隆得到了家蚕hnRNPA1基因的全长cDNA片段,其开放阅读框(ORF)序列为951 bp,编码316个氨基酸,预测分子量为34.98 kDa,等电点为5.15。编码蛋白在第18～90个氨基酸和109～181个氨基酸处为保守的RRM结构域。系统进化分析显示,家蚕hnRNPA1与小菜蛾hnRNPA1的亲缘关系最近。QRT-PCR结果显示,BmhnRNPA1在家蚕幼虫和蛹期的翅原基组织中均有表达,且在蛹期第3天的表达量达到峰值。Western blot进一步证实了实验结果。免疫组化分析结果显示,该蛋白存在于翅原基组织中,并定位于细胞核内。家蚕BmhRNPA1具有两个RNA结合结构域,属于hnRNPs家族,定位于细胞核内,表明其可能参与mRNA的选择性剪接作用。本研究结果为进一步探索该基因的功能提供了基础。
hnRNPA1(heterogeneous protein A1) is an important RNA binding protein. This study is to obtain full length cDNA of hnRNPA1 and to analyze its expression and localization in the wing discs of Bombyx mori. BmhnRNPA1 cDNA was cloned by reverse-transcription PCR(RT-PCR) using mRNA that was isolated from the larvae and its sequence was analyzed. The protein expression vector of pET32 a-BmhnRNPA1 was constructed and the recombinant protein was expressed and purified. Polyclonal antibody was generated. The quantitative real-time PCR(qRT-PCR), western blot and immunohistochemistry methods were used to analyze the expression and localization of BmhnRNPA1 in the wing discs during the larvae and pupa. The open reading frame of the hnRNPA1 cDNA was 951 bp in length and encoded 316 amino acids. The predicted molecular mass of the protein was 34.98 kDa and the isoelectric point was 5.15. The protein contains two conserved RRM domains between 18 and 90 amino acids and between 109 and 181 amino acids. Phylogenetic analysis showed that the BmhnRNPA1 was closely related to Plutella xylostella hnRNPA1. The results of QRT-PCR showed that BmhnRNPA1 expressed in the wing discs during the larval and pupal stage, and the expression level reached the peak on the 3~(rd) day of the pupal stage. The results of western blot confirmed the above results. Immunohistochemistry analysis showed that hnRNPA1 expressed in the wing disc and the protein was localized in the nuclei of wing disc cell. This study revealed that BmhRNPA1 has two RNA binding domains, belonging to the hnRNPs family and probably involving in the splicing of premature mRNA. This study provides a foundation for the function analysis of this protein.
hnRNPA1(heterogeneous protein A1) is an important RNA binding protein. This study is to obtain full length cDNA of hnRNPA1 and to analyze its expression and localization in the wing discs of Bombyx mori. BmhnRNPA1 cDNA was cloned by reverse-transcription PCR(RT-PCR) using mRNA that was isolated from the larvae and its sequence was analyzed. The protein expression vector of pET32 a-BmhnRNPA1 was constructed and the recombinant protein was expressed and purified. Polyclonal antibody was generated. The quantitative real-time PCR(qRT-PCR), western blot and immunohistochemistry methods were used to analyze the expression and localization of BmhnRNPA1 in the wing discs during the larvae and pupa. The open reading frame of the hnRNPA1 cDNA was 951 bp in length and encoded 316 amino acids. The predicted molecular mass of the protein was 34.98 kDa and the isoelectric point was 5.15. The protein contains two conserved RRM domains between 18 and 90 amino acids and between 109 and 181 amino acids. Phylogenetic analysis showed that the BmhnRNPA1 was closely related to Plutella xylostella hnRNPA1. The results of QRT-PCR showed that BmhnRNPA1 expressed in the wing discs during the larval and pupal stage, and the expression level reached the peak on the 3~(rd) day of the pupal stage. The results of western blot confirmed the above results. Immunohistochemistry analysis showed that hnRNPA1 expressed in the wing disc and the protein was localized in the nuclei of wing disc cell. This study revealed that BmhRNPA1 has two RNA binding domains, belonging to the hnRNPs family and probably involving in the splicing of premature mRNA. This study provides a foundation for the function analysis of this protein.
引文
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Guil S, Cáceres JF. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a [J].Nature Structural & Molecular Biology, 2007, 14 (7): 591.
Guo R, Li Y, Ning J,et al. HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells [J]. PLoS ONE, 2013, 8 (4): e62729.
Haynes SR, Johnson D, Raychaudhuri G,et al. The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group [J]. Nucleic Acids Research, 1991, 19 (1): 25-31.
Hovemann BT, Reim I, Werner S,et al. The protein Hrb57A of Drosophila melanogaster, closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins [J]. Gene, 2000, 245 (1): 127-137.
Huang M, Rech JE, Northington SJ,et al. The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles [J]. Molecular & Cellular Biology, 1994, 14 (1): 518-33.
Kamma H, Portman DG. Cell type-specific expression of hnRNP proteins [J].Experimental Cell Research, 1995, 221 (1): 187-196.
Matunis MJ, Matunis EL, Dreyfuss G. Isolation of hnRNP complexes from Drosophila melanogaster [J]. Journal of Cell Biology, 1992, 116 (2): 245.
Martinez-contreras R, Cloutier P, Shkreta L,et al. HnRNP proteins and splicing control [J]. Advances in Experimental Medicine & Biology, 2007, 623 (1): 123.
Mayeda A, Munroe SH, Cáceres JF, et al. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins [J]. Embo Journal, 1994, 13 (22): 5483.
Mayeda A, Krainer AR. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2 [J].Cell, 1992, 68 (2): 365.
Mayeda A, Munroe SH, Cáceres JF,et al. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins [J]. Embo Journal, 1994, 13 (22): 5483.
Mcafee JG, Shahied-Milam L, Soltaninassab SR,et al. A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain [J]. RNA, 1996, 2 (11): 1139.
Piňolroma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus andcytoplasm [J].Nature, 1992, 355 (6362): 730-732.
Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server [J].Nucleic Acids Research, 2014, 42 (Web Server issue): W320.
Shay AJW. A model for heterogeneous nuclear ribonucleoproteins in telomere and telomerase regulation [J].Oncogene, 2002, 21 (4): 580-583.
Sofola OA, Jin P, Qin Y,et al. RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS [J]. Neuron, 2007, 55 (4): 565-71.
Tingley WG, Roche KW, Thompson AK,et al. Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain [J]. Nature, 1993, 364 (6432): 70-73.
Burd CG, Dreyfuss G. RNA binding specificity of hnRNP A1: Significance of hnRNP A1 high‐affinity binding sites in pre-mRNA splicing [J].Embo Journal, 1994, 13 (5): 1197.
Birney E, Kumar S, Krainer AR. Analysis of the RNA-recognition motif and RS and RGG domains: Conservation in metazoan pre-mRNA splicing factors [J].Nucleic Acids Research, 1993, 21 (25): 5803.
Borah S, Wong AC, Steitz JA.Drosophila hnRNP A1 homologs Hrp36/ Hrp38 enhance U2-type versus U12-type splicing to regulate alternative splicing of the prospero twintron [J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106 (8): 2577-82.
Brooks AN, Duff MO, May G,et al. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins [J]. Genome Research, 2015, 25 (11): 1771.
Domsic JK, Wang Y, Mayeda A,et al. Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts [J]. Molecular & Cellular Biology, 2003, 23 (23): 8762-8772.
Dreyfuss G, Kim VN, Kataoka N. Messenger-RNA-binding proteins and the messages they carry [J].Nature Reviews Molecular Cell Biology, 2002, 3 (3): 195.
Dreyfuss G, Matunis MJ, Piňolroma S,et al. hnRNP proteins and the biogenesis of mRNA [J]. Annual Review of Biochemistry, 1993, 62 (1): 289.
Ding J, Hayashi MK, Zhang Y,et al. Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA [J]. Genes & Development, 1999, 13 (9): 1102-15.
Feng LC, Shen WD. Silkworm Anatomy and Physiology [M]. Beijing:Higher Education Press, 2015. [冯丽春, 沈卫德. 蚕体解剖生理学 [M]. 北京:高等教育出版社, 2015]
Geuens T, Bouhy D, Timmerman V. The hnRNP family: Insights into their role in health and disease [J].Human Genetics, 2016, 135 (8): 851.
Guil S, Cáceres JF. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a [J].Nature Structural & Molecular Biology, 2007, 14 (7): 591.
Guo R, Li Y, Ning J,et al. HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells [J]. PLoS ONE, 2013, 8 (4): e62729.
Haynes SR, Johnson D, Raychaudhuri G,et al. The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group [J]. Nucleic Acids Research, 1991, 19 (1): 25-31.
Hovemann BT, Reim I, Werner S,et al. The protein Hrb57A of Drosophila melanogaster, closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins [J]. Gene, 2000, 245 (1): 127-137.
Huang M, Rech JE, Northington SJ,et al. The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles [J]. Molecular & Cellular Biology, 1994, 14 (1): 518-33.
Kamma H, Portman DG. Cell type-specific expression of hnRNP proteins [J].Experimental Cell Research, 1995, 221 (1): 187-196.
Matunis MJ, Matunis EL, Dreyfuss G. Isolation of hnRNP complexes from Drosophila melanogaster [J]. Journal of Cell Biology, 1992, 116 (2): 245.
Martinez-contreras R, Cloutier P, Shkreta L,et al. HnRNP proteins and splicing control [J]. Advances in Experimental Medicine & Biology, 2007, 623 (1): 123.
Mayeda A, Munroe SH, Cáceres JF, et al. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins [J]. Embo Journal, 1994, 13 (22): 5483.
Mayeda A, Krainer AR. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2 [J].Cell, 1992, 68 (2): 365.
Mayeda A, Munroe SH, Cáceres JF,et al. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins [J]. Embo Journal, 1994, 13 (22): 5483.
Mcafee JG, Shahied-Milam L, Soltaninassab SR,et al. A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain [J]. RNA, 1996, 2 (11): 1139.
Piňolroma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus andcytoplasm [J].Nature, 1992, 355 (6362): 730-732.
Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server [J].Nucleic Acids Research, 2014, 42 (Web Server issue): W320.
Shay AJW. A model for heterogeneous nuclear ribonucleoproteins in telomere and telomerase regulation [J].Oncogene, 2002, 21 (4): 580-583.
Sofola OA, Jin P, Qin Y,et al. RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS [J]. Neuron, 2007, 55 (4): 565-71.
Tingley WG, Roche KW, Thompson AK,et al. Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain [J]. Nature, 1993, 364 (6432): 70-73.