猪hnRNPUL1基因克隆及变异剪接体鉴定

doi:10.11843/j.issn.0366-6964.2020.03.005

摘要/Abstract

摘要： 旨在克隆民猪hnRNPUL1基因全长编码区（complete coding sequence，CDS）序列并进行可变剪接分析，研究其组织表达和亚细胞定位情况。本研究以3头3月龄民猪为试验材料，利用RT-PCR技术克隆hnRNPUL1基因CDS及可变剪接体，应用qRT-PCR检测其在心、肝、脾、肺、肾、胃、大肠、小肠、背肌、腿肌等组织中的表达情况，同时，在生物信息学预测的基础上，通过融合表达绿色荧光蛋白的方法鉴定核定位序列。研究结果表明，猪hnRNPUL1基因（GenBank accession No.：MN399154）CDS全长2 580 bp，预期编码的多肽链存在着hnRNPs家族的特征性结构域——SAP、SPRY和AAA；猪hnRNPUL1普遍表达于所检测的各组织中，其中在脾脏中表达量最高，在腿肌中表达量最低；核定位序列（NLS）位于多肽链的133~430 aa处，与生物学信息学预测的结果存在着偏差；同时获得了2个变异剪接体和11种可变剪接片段。本研究结果对进一步揭示猪hnRNPUL1基因功能及转录调控机制提供了基础。

关键词: 猪, hnRNPUL1, 克隆, 变异剪接体

Abstract: The study was conducted to clone the complete coding sequence (CDS) and transcript variants of porcine hnRNPUL1, and to analyze its tissue expression and subcellular localization. In this study, three 3-month-old Min pigs were used. The CDS and transcript variants of porcine hnRNPUL1 were cloned using RT-PCR method, and the expression was detected in tissues including heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, back muscle and leg muscle by qRT-PCR. At the same time, the subcellular localization was analyzed by inserting the fragments of CDS into pEGFP-N1 vector on the basis of bioinformatics prediction. It was found that the CDS of porcine hnRNPUL1 gene (GenBank accession No.:MN399154) was 2 580 bp of length, and the predicted polypeptide contained SAP, SPRY, and AAA domains, the characteristic domains of hnRNPs family. Porcine hnRNPUL1 was ubiquitously expressed in all detected tissues, with the highest level in the spleen but the lowest in leg muscle. The nuclear localization sequence(NLS) was located at 133-430 aa of the polypeptide chain, which was different from the result predicted by the bioinformatics methods. In addition, 2 splice variants and 11 alternative splicing fragments were identified. The results have provided the basis for further revealing the function and transcriptional regulation mechanism of porcine hnRNPUL1.

Key words: pig, hnRNPUL1, cloning, splice variants

中图分类号:

S828.2

宋艳芳, 张彩霞, 杜芳芳, 马骏杰, 刘东宇, 刘娣, 杨秀芹. 猪hnRNPUL1基因克隆及变异剪接体鉴定[J]. 畜牧兽医学报, 2020, 51(3): 443-451.

SONG Yanfang, ZHANG Caixia, DU Fangfang, MA Junjie, LIU Dongyu, LIU Di, YANG Xiuqin. Cloning and Identification of Splice Variants of the Porcine hnRNPUL1 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 443-451.

参考文献 45

[1]	DREYFUSS G,KIM V N,KATAOKA N.Messenger-RNA-binding proteins and the messages they carry[J].Nat Rev Mol Cell Biol,2002,3(3):195-205.
[2]	ROSHON M J,RULEY H E.Hypomorphic mutation in hnRNP U results in post-implantation lethality[J].Transgenic Res,2005, 14(2):179-192.
[3]	GEUENS T,BOUHY D,TIMMERMAN V.The hnRNP family:insights into their role in health and disease[J].Hum Genet, 2016,135(8):851-867.
[4]	CARPENTER B,MACKAY C,ALNABULSI A,et al.The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression[J].Biochim Biophys Acta,2006,1765(2):85-100.
[5]	KRECIC A M,SWANSON M S.hnRNP complexes:composition,structure,and function[J].Curr Opin Cell Biol,1999, 11(3):363-371.
[6]	GABLER S,SCHVTT H,GROITL P,et al.E1B 55-kilodalton-associated protein:a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs[J].J Virol,1998,72(10):7960-7971.
[7]	BACHI A,BRAUN I C,RODRIGUES J P,et al.The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates[J].RNA,2000,6(1):136-158.
[8]	BARRAL P M,RUSCH A,TURNELL A S,et al.The interaction of the hnRNP family member E1B-AP5 with p53[J].FEBS Lett,2005,579(13):2752-2758.
[9]	POLO S E,BLACKFORD A N,CHAPMAN J R,et al.Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair[J].Mol Cell,2012,45(4):505-516.
[10]	SCHREIBER V,DANTZER F,AME J C,et al.Poly (ADP ribose):novel functions for an old molecule[J].Nat Rev Mol Cell Biol,2006,7(7):517-528.
[11]	SHIFFMAN D,ROWLAND C M,LOUIE J Z,et al.Gene variants of VAMP8 and HNRPUL1 are associated with early-onset myocardial infarction[J].Arterioscler Thromb Vasc Biol,2006,26(7):1613-1618.
[12]	CHATTERJEE N,WALKER G C.Mechanisms of DNA damage,repair,and mutagenesis[J].Environ Mol Mutagen,2017, 58(5):235-263.
[13]	HONG Z H,JIANG J,MA J L,et al.The role of hnRPUL1 involved in DNA damage response is related to PARP1[J].PLoS One,2013,8(4):e60208.
[14]	JI Y X,WANG Q,ZHAO Q,et al.Autophagy suppression enhances DNA damage and cell death upon treatment with PARP inhibitor Niraparib in laryngeal squamous cell carcinoma[J].Appl Microbiol Biotechnol,2019,103(23-24):9557-9568.
[15]	MACCIÒ A,MADEDDU C.The mechanism of cancer cell death by PARP inhibitors goes beyond DNA damage alone[J].Int J Cancer,2019,145(9):2594-2596.
[16]	CHABANON R M,SORIA J C,LORD C J,et al.Beyond DNA repair:the novel immunological potential of PARP inhibitors[J]. Mol Cell Oncol,2019,6(2):1585170.
[17]	VISCONTI R,GRIECO D.New insights on oxidative stress in cancer[J].Curr Opin Drug Discov Devel,2009,12(2):240-245.
[18]	REUTER S,GUPTA S C,CHATURVEDI M M,et al.Oxidative stress,inflammation,and cancer:how are they linked?[J].Free Radic Biol Med,2010,49(11):1603-1616.
[19]	SHARMA A,JOHNSON A.Exosome DNA:critical regulator of tumor immunity and a diagnostic biomarker[J].J Cell Physiol, 2020, 235(3):1921-1932.
[20]	NAYLER O,STRÄTLING W,BOURQUIN J P,et al.SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements[J].Nucleic Acids Res,1998,26(15):3542-3549.
[21]	ARAVIND L,KOONIN E V.SAP-a putative DNA-binding motif involved in chromosomal organization[J].Trends Biochem Sci,2000,25(3):112-114.
[22]	YAP M W,NISOLE S,STOYE J P.A single amino acid change in the SPRY domain of human Trim5α leads to HIV-1 restriction[J]. Curr Biol,2005,15(1):73-78.
[23]	RHODES D A,DE BONO B,TROWSDALE J.Relationship between SPRY and B30.2 protein domains.Evolution of a component of immune defence?[J].Immunology,2005,116(4):411-417.
[24]	OGURA T,WILKINSON A J.AAA+ superfamily ATPases:common structure——diverse function[J].Genes Cells,2001,6(7):575-597.
[25]	MISTELI T,SPECTOR D L.Applications of the green fluorescent protein in cell biology and biotechnology[J].Nat Biotechnol, 1997,15(10):961-964.
[26]	张霞,宋雪,王配,等.版纳微型猪近交系CMAH基因克隆及亚细胞定位研究[J].河北农业大学学报,2019,42(3):94-98.ZHANG X,SONG X,WANG P,et al.Cloning and subcellular location of CMAH gene in Banna mini-pig inbred line[J].Journal of Hebei Agricultural University,2019,42(3):94-98.(in Chinese)
[27]	范兴兴,李新建,李改英,等.猪Fosl2基因克隆、结构预测及其mRNA发育性表达变化[J].畜牧兽医学报,2015,46(2):211-218.FAN X X,LI X J,LI G Y,et al.Gene cloning,bioinformatics analysis and developmental expression of Fosl2 gene in pig[J].Acta Veterinaria et Zootechnica Sinica,2015,46(2):211-218.(in Chinese)
[28]	赵为民,涂枫,方晓敏,等.猪PKM2基因的序列分析与组织表达及亚细胞定位[J].湖南农业大学学报:自然科学版,2019,45(1):68-77.ZHAO W M,TU F,FANG X M,et al.Sequence analysis and tissue expression and subcellular localization of porcine PKM2 gene[J].Journal of Hunan Agricultural University:Natural Sciences,2019,45(1):68-77.(in Chinese)
[29]	CAPPELLI S,ROMANO M,BURATTI E.Systematic analysis of gene expression profiles controlled by hnRNP Q and hnRNP R,two closely related human RNA binding proteins implicated in mRNA processing mechanisms[J].Front Mol Biosci,2018,5:79.
[30]	GASPERINI L,ROSSI A,CORNELLA N,et al.The hnRNP RALY regulates PRMT1 expression and interacts with the ALS-linked protein FUS:implication for reciprocal cellular localization[J].Mol Biol Cell,2018,29(26):3067-3081.
[31]	RUAN Q T,YAZDANI N,BLUM B C,et al.A mutation in Hnrnph1 that decreases methamphetamine-induced reinforcement,reward,and dopamine release and increases synaptosomal hnRNP H and mitochondrial proteins[J].J Neurosci, 2019, 11:1808-1819.
[32]	IZUMI H,FUNA K.Telomere function and the G-Quadruplex formation are regulated by hnRNP U[J].Cells,2019,8(5):390.
[33]	NAGASAKI H,ARITA M,NISHIZAWA T,et al.Automated classification of alternative splicing and transcriptional initiation and construction of visual database of classified patterns[J].Bioinformatics,2006,22(10):1211-1216.
[34]	PAN Q,SHAI O,LEE L J,et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[J].Nat Genet,2008,40(12):1413-1415.
[35]	邵红伟,张文峰,胡青莲,等.两种存活蛋白变异剪接体在HeLa细胞中的表达定位及相互作用[J].中国生物化学与分子生物学报,2009,25(12):1131-1136.SHAO H W,ZHANG W F,HU Q L,et al.Expression and location of two survivin splice variants and their interaction in HeLa Cells[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(12):1131-1136.(in Chinese)
[36]	赵晋枫,武宝爱.有氧运动影响低密度脂蛋白受体pre-mRNA的选择性剪接及组蛋白修饰机制研究[J].山东体育学院学报,2018,34(6):78-83.ZHAO J F,WU B A.Histone modifications involved in alternative splicing of LDLR pre-mRNA and the influence of aerobic exercise[J].Journal of Shandong Sport University,2018,34(6):78-83.(in Chinese)
[37]	张天,李祥龙,周荣艳,等.不同毛色山羊皮肤组织Agouti基因剪接体类型研究[J].畜牧兽医学报,2015,46(11):1934-1943.ZHANG T,LI X L,ZHOU R Y,et al.Study on Agouti spliceosome types in goat skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1934-1943.(in Chinese)
[38]	李利族,韩丽鑫,王金奎,等.猪PILRA基因克隆及变异剪接体鉴定[J].遗传,2015,37(9):926-931.LI L Z,HAN L X,WANG J K,et al.Cloning and identification of splice variants of the porcine PILRA gene[J].Hereditas (Beijing),2015,37(9):926-931.(in Chinese)
[39]	NUTTER C A,BUBENIK J L,OLIVEIRA R,et al.Cell-type-specific dysregulation of RNA alternative splicing in short tandem repeat mouse knockin models of myotonic dystrophy[J].Genes Dev,2019,33(23-24):1635-1640.
[40]	谢玉霜,包永芬,白育庭,等.变异剪接体的结构、功能及其在疾病治疗中的应用[J].生命的化学,2019,39(4):660-664.XIE Y S,BAO Y F,BAI Y T,et al.The structure and function of the spliceosome and its application in the diseases therapy[J].Chemistry of Life,2019,39(4):660-664.(in Chinese)
[41]	JIA R,AJIRO M,YU L L,et al.Oncogenic splicing factor SRSF3 regulates ILF3 alternative splicing to promote cancer cell proliferation and transformation[J].RNA,2019,25(5):630-644.
[42]	BRAGGIN J E,BUCKS S A,COURSE M M,et al.Alternative splicing in a presenilin 2 variant associated with Alzheimer disease[J].Ann Clin Trans Neurol,2019,6(4):762-777.
[43]	WU ZE,LIANG J H,WANG C P,et al.Alternative splicing provides a mechanism to regulate LlHSFA3 function in response to heat stress in lily[J].Plant Physiol,2019,181(4):1651-1667.
[44]	LI L P,YAN S X,ZHANG H,et al.Interaction of hnRNP K with MAP 1B-LC1 promotes TGF-β1-mediated epithelial to mesenchymal transition in lung cancer cells[J].BMC Cancer,2019,19(1):894.
[45]	LIANG S Z,YU B,QIAN D M,et al.Human cytomegalovirus ie2 affects the migration of glioblastoma by mediating the different splicing patterns of RON through hnRNP A2B1[J].Neuroreport,2019,30(12):805-811.