RBM15结合促进RNA内含子或外显子滞留

RBM15是一种RNA结合蛋白,参与到RNA的m6A修饰及可变剪接调控中.然而,RBM15在转录组水平如何调控可变剪接尚不清楚.本研究应用超分辨率荧光显微镜技术发现,RBM15在细胞核中形成斑点状结构,且与核斑有密切接触或完全定位于核斑中.核斑为细胞核中无膜细胞器,富含多种剪接因子,这提示RBM15可能参与到可变剪接的...     

NDRG2 基因六种可变剪接体的分型鉴定

目的:抑癌基因N-myc下游调节基因2(N-myc down stream regulated gene 2,NDRG 2)在抑制肿瘤的发生及进展过程中具有重要作用,本研究旨在区分并鉴定NDRG2基因mRNA的6种可变剪接体。方法:针对NDRG2基因mRNA6种剪接体外显子的差别设计6对特异性引物。分别从正常人脑组织和胶质瘤细胞U251中提取总RNA,反转录为cDNA,利用高保真酶扩增目的剪接体DNA,并根据PCR扩增产物电泳条带的有无和大小初步判断剪接体的型别,最后将PCR产物胶回收进行测序鉴定。结果:利用本实验中设计的引物可以将人脑组织和U251胶质瘤细胞中的NDRG 2基因mRNA剪接体经行分型鉴定,确定了分型引物的可行性。人脑组织和U251细胞中均含有A、B、D和E四种剪接体。结论:利用本研究设计的特异性引物进行PCR扩增,可以判断某种细胞或组织中表达的NDRG2mRNA分子剪接体的型别。有助于未来深入研究抑癌基因NDRG2在肿瘤发生发展中的抑制作用与不同剪接体的表达相关性。     

RBM6通过GSK-3β/β-catenin调节途径对膀胱尿路上皮癌细胞发挥抗增殖和促进凋亡作用

RNA结合模体蛋白(RBM)家族几乎在所有细胞中均有表达,而且在进化上高度保守。以前的研究表明,RBM5在膀胱尿路上皮癌(bladder urothelial carcinoma,BUC)组织中表达下调,由此引起的细胞凋亡减少,进而促进肿瘤的进展。然而,RBM5氨基酸序列与其同源的RBM6在膀胱尿路上皮癌中的作用及相关机制尚不清楚。本研究检测RBM6在膀胱尿路上皮癌中的表达,探索其在膀胱癌细胞系中过表达对细胞增殖和凋亡的影响。利用qRT-PCR和Western印迹等技术,研究发现,RBM6在人膀胱尿路上皮癌组织和所检测的2个膀胱癌细胞系中表达均显著下调,并且其下调程度与患者的预后不良呈正相关。MTS检测细胞增殖的结果显示,RBM6过表达导致细胞增殖活性下降。细胞克隆形成实验结果也表明,RBM6过表达抑制细胞克隆形成能力(P0.01)。原位末端转移酶标记技术(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, TUNEL染色)检测细胞凋亡表明,在RBM6过表达的细胞中,TUNEL阳性细胞数由对照组的17.17±3.27增加到44.00±8.04(P0.05)。RBM6抗增殖及促凋亡的作用机制研究发现,在T24细胞中过表达RBM6后,β-联蛋白(β-catenin)和G_1/S-特异性细胞周期蛋白-D1(G_1/S-specific cyclin-D1,cyclin D1)mRNA表达水平分别降低65%和79%,对应的蛋白质水平分别降低60%和73%;反之,细胞周期蛋白依赖性激酶抑制因子1A(cyclin dependent kinase inhibitor 1A,CDKN1A/p21)mRNA表达水平升高约1.9倍,对应蛋白质水平升高约1.8倍。重要的是,糖原合酶激酶3 beta(glycogen synthase kinase-3 beta,GSK-3β)磷酸化水平升高约2.4倍。细胞过表达β-联蛋白促进细胞增殖(P0.05),而利用小分子化合物LY294002促进GSK-3β磷酸化后,抑制细胞增殖(P0.05)。综上所述,RBM6通过GSK-3β/β-catenin调节途径对膀胱尿路上皮癌细胞发挥抗增殖和促进凋亡作用。干预该调节途径可能是治疗膀胱尿路上皮癌的潜在靶点。     

RNA结合基序蛋白3的生物学功能

RNA结合基序蛋白3(RNA binding motif protein 3,RBM3)是在哺乳动物细胞中发现的重要冷应激蛋白,伴随冷应激过程其表达量上调。研究发现RBM3与冷诱导RNA结合蛋白(cold inducible RNA binding protein,CIRP)在核酸结构和氨基酸结构上高度保守,具有较高的同源性,且发挥作用的方式也有很多相似之处。RBM3不仅具有介导冷诱导下细胞抗凋亡的保护作用,还能参与人和动物的缺氧应激、缺血应激、紫外照射应激、细胞增殖、骨骼肌调节、生殖发育,疾病的治疗等多种生理过程,而且它还能够作为原癌基因以及参与小RNA(microRNA,简称miRNA)生物学过程的调控作用。因此,深入开展RBM3功能性研究和应用性研究具有重要的基础研究价值和临床研究意义。     

草鱼RBM cDNA克隆及序列分析

目的:克隆草鱼RNA剪切蛋白(RNA binding motif protein-RBM)基因.方法:采用RT -PCR技术从草鱼性腺总RNA中克隆RBM基因,将其插入pBS-T载体上,经菌液PCR鉴定后进行 测序并对测序结果进行生物信息分析.结果:获得草鱼RBM cDNA部分序列 ,大小为384bp,预 测编码127个氨基酸残基;RBM基因进化与物种形态进化类似;不同物种间RBM基因一级结构 保守性不高,但氨基酸序列保守性较高,且存在高度保守的氨基酸位点.结论: 成功地克隆了草鱼RBM cDNA部分序列,为获RBM cDNA全长序列及后续研究提供理论基础.     

Prp8蛋白在前体mRNA剪接中的作用

前体mRNA的剪接是基因表达的关键一步,发生在蛋白质的转录之后与合成之前.在前体mRNA剪接加工过程中需要将转录本中的内含子切除,因为它会干扰基因的转录.前体mRNA的剪接发生在细胞核中,是在一个大的RNA与蛋白质的复合物即剪接体的催化下完成的.Prp8 （precursor mRNA processing）是参与前体mRNA剪接的最大的蛋白,其序列从酵母到人类是高度保守的.Prp8同时也是细胞核内一个最重要的剪接因子.在剪接过程中,Prp8组成剪接体的催化中心.有人推断Prp8是剪接体的支架蛋白,很可能在催化中心起到锚定RNA的作用,同时也调节着激活剪接体所必需的构象变化.Prp8还与色素性视网膜炎的发生密切相关.     

RNA结合模体蛋白3对细胞总蛋白质合成的影响及其靶基因的鉴定

RNA结合模体蛋白3 (RNA binding motif protein 3,RBM3)受低温应激产生,参与介导亚低温的神经保护功能,但其作用机制及其下游靶分子尚不清楚。本研究构建了人RBM3基因的重组表达质粒,运用一种新型的、非放射性方法即翻译表面感应(surface sensing of translation,SUnSET)来检测RBM3过表达对细胞总蛋白质合成(global protein synthesis,GPS)的影响。结果显示,RBM3过表达使细胞总蛋白质的合成水平上调23. 7%(P 0. 001),这与RBM3过表达引发的真核翻译延伸因子2 (eukaryotic translation elongation factor 2,e EF2)及真核翻译起始因子2α(eukaryotic initiation factor 2α,eIF2α)的活性增高相一致。对RBM3可能的下游靶基因内质网蛋白3(reticulon 3,RTN3),以及Yes相关蛋白1 (Yes-associated protein 1,YAP1)的表达进行分析。结果显示,RBM3使RTN3及YAP1在蛋白质水平的表达分别提高了51. 7%(P 0. 01)与43. 3%(P 0. 01)。与蛋白质水平变化相比,RBM3使YAP1在mRNA水平上调了2. 0倍(P 0. 001),但对RTN3的mRNA表达未见显著影响。以上研究表明,SUnSET是一种稳定、可靠的细胞GPS的检测手段; RBM3可显著促进细胞GPS,且对其下游基因RTN3和YAP1存在靶向关系。本研究的结果为深入探讨RBM3的神经保护作用机制提供了理论基础。     

RBM10通过影响miR-21稳定性参与调控人乳腺癌细胞增殖及转移活性

目的:分析miR-21和RNA结合基序(RBM)在人乳腺癌组织及相应肿瘤细胞中的表达相关性和功能联系。方法:通过Oncomine或starBase数据库分析miR-21和RBM10表达数据,揭示其在人乳腺癌和对照组织中的表达差异;采用人乳腺癌细胞MCF-7、MDA-MB-231和永生化的人乳腺正常上皮细胞MCF-10A进一步分析miR-21和RBM10在乳腺癌细胞和正常乳腺细胞中的表达差异;采用CCK-8法检测敲低RBM10对MCF-7细胞增殖的影响;采用Transwell实验检测敲低RBM10对MCF-7细胞侵袭与迁移能力的调控差异;用放线菌素D处理MCF-7细胞,检测不同时间点miR-21的相对含量反映其稳定性。结果:生物信息学分析结果显示miR-21和RBM10在乳腺癌组织中显著高表达;与组织表达结果类似,在乳腺癌细胞中miR-21和RBM10的表达量显著高于正常乳腺细胞;miR-21与RBM10的表达呈正相关;敲低RBM10抑制MCF-7细胞的增殖、迁移与侵袭,而同时过表达miR-21会减弱这种抑制效应;敲低RBM10能够显著降低miR-21的稳定性。结论:RBM10通过影响miR-21的稳定性参与调控人乳腺癌细胞功能活性。RBM10在肿瘤中的作用仍有待进一步研究,不能简单地概括为一种抑癌因子或致癌因子。     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

肝素酶基因可变剪接体的克隆及鉴定

目的:克隆肝素酶基因的可变剪接体并测序。方法:根据人肝素酶的cDNA序列设计引物,用RT-PCR方法从正常人外周血白细胞中扩增肝素酶基因的可变剪接体,构建至pGEM-T Easy载体中,转化大肠杆菌DH5α感受态细胞,筛选阳性克隆并进行序列测定。结果:获得了肝素酶基因的3种可变剪接体形式,即5号外显子缺失可变剪接体、6号外显子缺失可变剪接体、5和6号外显子缺失可变剪接体,其中后2种可变剪接体尚未报道。结论:克隆了肝素酶基因的3种可变剪接体,有助于研究各种肝素酶可变剪接体编码蛋白的结构和功能及其在肿瘤发生转移过程中的作用。     

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.     

The tumor suppressor gene RBM5 inhibits lung adenocarcinoma cell growth and induces apoptosis

ABSTRACT: BACKGROUND: The loss of tumor suppressor gene (TSG) function is a critical step in the pathogenesis of human lung cancer. RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 demonstrated tumor suppressor activity. However, the role of RBM5 played in the occurrence and development of lung cancer is still not well understood. METHOD: Paired non-tumor and tumor tissues were obtained from 30 adenocarcinomas. The expression of RBM5 mRNA and protein was examined by RT-PCR and Western blot. A549 cell line was used to determine the apoptotic function of RBM5 in vitro. A549 cells were transiently transfected with pcDNA3.1-RBM5. AnnexinV analysis was performed by flow cytometry. Expression of Bcl-2, cleaved caspase-3, caspase-9 and PAPP proteins in A549 lung cancer cells and the A549 xenograft BALB/c nude mice model was determined by Western blot. Tumor suppressor activity of RBM5 was also examined in the A549 xenograft model treated with pcDNA3.1-RBM5 plasmid carried by attenuated Salmonella typhi Ty21a. Result The expression of RBM5 mRNA and protein was decreased significantly in adenocarcinoma tissues compared to that in the non-tumor tissues. In addition, as compared to the vector control, a significant growth inhibition of A549 lung cancer cells was observed when transfected with pcDNA3.1-RBM5 as determined by cell proliferation assay. We also found that overexpression of RBM5 induced both early and late apoptosis in A549 cells using AnnexinV/PI staining as determined by flow cytometry. Furthermore, the expression of Bcl-2 protein was decreased, whereas the expression of cleaved caspase-3, caspase-9 and PARP proteins was significantly increased in the RBM5 transfected cells; similarly, expression of decreased Bcl-2 and increased cleaved caspase-3 proteins was also examined in the A549 xenograft model. More importantly, we showed that accumulative and stable overexpression of RBM5 in the A549 xenograft BALB/c nude mice model significantly inhibited the tumor growth rate in vivo as compared to that in the control. CONCLUSION: Our study demonstrates that RBM5 can inhibit the growth of lung cancer cells and induce apoptosis both in vitro and in vivo, which suggests that RBM5 might be used as a potential biomarker or target for lung cancer diagnosis and chemotherapy. Moreover, we propose a novel animal model set up in BALB/c nude mice treated with attenuated Salmonella as a vector carrying plasmids to determine RBM5 function in vivo.     

RNA binding motif (RBM) proteins: A novel family of apoptosis modulators?

RBM5 is a known modulator of apoptosis, an RNA binding protein, and a putative tumor suppressor. Originally identified as LUCA-15, and subsequently as H37, it was designated "RBM" (for RNA Binding Motif) due to the presence of two RRM (RNA Recognition Motif) domains within the protein coding sequence. Recently, a number of proteins have been attributed with this same RBM designation, based on the presence of one or more RRM consensus sequences. One such protein, RBM3, was also recently found to have apoptotic modulatory capabilities. The high sequence homology at the amino acid level between RBM5, RBM6, and particularly, RBM10 suggests that they, too, may play an important role in regulating apoptosis. It is the intent of this article to ammalgamate the data on the ten originally identified RBM proteins in order to question the existence of a novel family of RNA binding apoptosis regulators.     

miR‐938 promotes cell proliferation by regulating RBM5 in lung adenocarcinoma cells

A growing body of research suggests that microRNAs (miRNAs) may play a key part in the progression of various cancers, including lung adenocarcinoma (LUAD). However, the expression and mechanism of miR‐938 (microRNA‐938) in LUAD have not been defined. Compared with adjacent tissues, the level of miR‐938 was up‐regulated in LUAD tissues. miR‐938 expression was significantly associated with tumor size. In vitro assays indicated that miR‐938 expression was also increased in the LUAD cell lines. Overexpression of miR‐938 promoted LUAD cell proliferation, whereas down‐regulation of miR‐938 had the opposite effect. We identified RNA‐binding protein 5 (RBM5) as a potential target gene of miR‐938 in LUAD. Expression of RBM5 was down‐regulated in LUAD tumor tissues and negatively correlated with expression of miR‐938. Up‐regulation of RBM5 reversed cell proliferation by inhibition of miR‐938 expression in LUAD cells. These results showed that miR‐938 may act as an oncogenic miRNA by targeting RBM5 in LUAD, indicating that miR‐938 could be used as a potential therapeutic target for LUAD patients.     

The 3p21.3 tumor suppressor RBM5 resensitizes cisplatin-resistant human non-small cell lung cancer cells to cisplatin

Objective: Increasing RBM5 levels inhibit tumor cell growth and promote apoptosis. In this study, we investigated the role of RBM5 in the cisplatin resistance observed in human lung non-small cell lung cancer cells and evaluated the effect of RBM5 modulation on cell growth inhibition and apoptosis induced by cisplatin in the parental non-small cell lung cancer cells A549 and their cisplatin resistant counterparts, A549/DDP cells. Methods: RBM5 mRNA and protein expression in the A549 and A549/DDP cells was analyzed by semi-quantitative RT-PCR and western blot. The A549/DDP cells were then transfected with a pcDNA3-RBM5 plasmid, and an RBM5-specific siRNA was transfected into A549 cells, prior to treatment with cisplatin. Semi-quantitative RT-PCR and western blot analyses were performed to confirm the expression of RBM5 mRNA or protein, and knockdown of RBM5 mRNA or protein, respectively. MTT assays were used to evaluate chemosensitivity to cisplatin. Apoptosis was assessed by DAPI nuclear staining and flow cytometric analysis with an Annexin-V-FITC apoptosis kit. Cytosolic cytochrome c, cleaved caspase-3 and cleaved caspase-9 were detected by western blot. Results: The expression of RBM5 mRNA and protein was significantly reduced in the A549/DDP cells compared with the A549 cells. Exogenous expression of RBM5 by the pcDNA3-RBM5 resensitized the response of A549/DDP to cisplatin, resulting in a significant increase in tumor-suppressing activity induced by cisplatin. In contrast, downregulation of RBM5 with siRNA in the A549 cells inhibited cisplatin-induced apoptosis. We also found that the RBM5-enhanced chemosensitivity was associated with the release of cytochrome c into the cytosol, activation of caspase-9 and the downstream marker caspase-3. Conclusion: Our results demonstrate that RBM5 may serve as a biomarker with the ability to predict a response to cisplatin. It may also act as a prognostic indicator in lung cancer patients. Our findings suggest that there may be clinical utility for ectopic RBM5 such as enhancing and resensitizing nonresponders to cisplatin.