这项工作是由美国国立卫生研究院资助R01-CA158283和国家自然科学基金委员会授予31400726到ZWYW由青年千人计划和中国国家自然科学基金（赠款31471235和81422038）的资助支持。 XY是由中国博士后科学基金会（2015M571612）资助。

<ol>
	<li>Wang, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456 (7221), 470-476 (2008).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nat Genet. 40 (12), 1413-1415 (2008).</li><li>Wang, Z., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+regulation:+from+a+parts+list+of+regulatory+elements+to+an+integrated+splicing+code.">Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.</a> Rna. 14 (5), 802-813 (2008).</li><li>Matera, A. G., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+day+in+the+life+of+the+spliceosome.">A day in the life of the spliceosome.</a> Nat Rev Mol Cell Biol. 15 (2), 108-121 (2014).</li><li>Singh, R. K., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-mRNA+splicing+in+disease+and+therapeutics.">Pre-mRNA splicing in disease and therapeutics.</a> Trends Mol Med. 18 (8), 472-482 (2012).</li><li>Wang, G. -. S., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+in+disease:+disruption+of+the+splicing+code+and+the+decoding+machinery.">Splicing in disease: disruption of the splicing code and the decoding machinery.</a> Nat Rev Genet. 8 (10), 749-761 (2007).</li><li>Li, Y. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+splicing+is+a+primary+link+between+genetic+variation+and+disease.">RNA splicing is a primary link between genetic variation and disease.</a> Science. 352 (6285), 600-604 (2016).</li><li>Scotti, M. M., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mis-splicing+in+disease.">RNA mis-splicing in disease.</a> Nat Rev Genet. 17 (1), 19-32 (2016).</li><li>Black, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+alternative+pre-messenger+RNA+splicing.">Mechanisms of alternative pre-messenger RNA splicing.</a> Annu Rev Biochem. 72 (1), 291-336 (2003).</li><li>Graveley, B. R., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine/serine-rich+domains+of+SR+proteins+can+function+as+activators+of+pre-mRNA+splicing.">Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing.</a> Mol cell. 1 (5), 765-771 (1998).</li><li>Del Gatto-Konczak, F., Olive, M., Gesnel, M. -. C., Breathnach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hnRNP+A1+recruited+to+an+exon+in+vivo+can+function+as+an+exon+splicing+silencer.">hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer.</a> Mol Cell Biol. 19 (1), 251-260 (1999).</li><li>Auweter, S. D., Oberstrass, F. C., Allain, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+binding+of+single-stranded+RNA:+is+there+a+code+for+recognition.">Sequence-specific binding of single-stranded RNA: is there a code for recognition.</a> Nucleic Acids Res. 34 (17), 4943-4959 (2006).</li><li>Dong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+modular+binding+code+for+cytosine+recognition+in+Pumilio/FBF+(PUF)+RNA-binding+domains.">Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.</a> J Biol Chem. 286 (30), 26732-26742 (2011).</li><li>Filipovska, A., Razif, M. F., Nyg&#229;rd, K. K., Rackham, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+code+for+RNA+recognition+by+PUF+proteins.">A universal code for RNA recognition by PUF proteins.</a> Nat Chem Biol. 7 (7), 425-427 (2011).</li><li>Wei, H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+RNA-binding+proteins+with+diverse+activities.">Engineering RNA-binding proteins with diverse activities.</a> Wiley Interdiscip Rev RNA. 6 (6), 597-613 (2015).</li><li>Wang, Y., Cheong, C. -. G., Hall, T. M. T., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+splicing+factors+with+designed+specificities.">Engineering splicing factors with designed specificities.</a> Nat Methods. 6 (11), 825-830 (2009).</li><li>Choudhury, R., Tsai, Y. S., Dominguez, D., Wang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+RNA+endonucleases+with+customized+sequence+specificities.">Engineering RNA endonucleases with customized sequence specificities.</a> Nat Commun. 3, 1147 (2012).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+network+of+factors+with+overlapping+affinities+represses+splicing+through+intronic+elements.">A complex network of factors with overlapping affinities represses splicing through intronic elements.</a> Nat Struct Mol Biol. 20 (1), 36-45 (2013).</li><li>McCabe, B. C., Gollnick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+levels+of+trp+RNA-binding+attenuation+protein+in+Bacillus+subtilis.">Cellular levels of trp RNA-binding attenuation protein in Bacillus subtilis.</a> J Bacteriol. 186 (15), 5157-5159 (2004).</li><li>Wang, Y., Ma, M., Xiao, X., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intronic+splicing+enhancers,+cognate+splicing+factors+and+context-dependent+regulation+rules.">Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules.</a> Nat Struct Mol Biol. 19 (10), 1044-1052 (2012).</li><li>Choudhury, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+splicing+activator+DAZAP1+integrates+splicing+control+into+MEK/Erk-regulated+cell+proliferation+and+migration.">The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration.</a> Nat Commun. 5, (2014).</li><li>Xiao, X., Wang, Z., Jang, M., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coevolutionary+networks+of+splicing+cis-regulatory+elements.">Coevolutionary networks of splicing cis-regulatory elements.</a> Proc Natl Acad Sci U S A. 104 (47), 18583-18588 (2007).</li><li>Wang, Z., Xiao, X., Van Nostrand, E., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+and+specific+functions+of+exonic+splicing+silencers+in+splicing+control.">General and specific functions of exonic splicing silencers in splicing control.</a> Mol Cell. 23 (1), 61-70 (2006).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1 (1), 241-245 (2006).</li><li>Venables, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+and+alternative+splicing+in+cancer.">Aberrant and alternative splicing in cancer.</a> Cancer Res. 64 (21), 7647-7654 (2004).</li><li>Cooke, A., Prigge, A., Opperman, L., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+translational+regulation+using+the+PUF+protein+family+scaffold.">Targeted translational regulation using the PUF protein family scaffold.</a> Proc Natl Acad Sci U S A. 108 (38), 15870-15875 (2011).</li><li>He, C., Zhou, F., Zuo, Z., Cheng, H., Zhou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+view+of+cancer-specific+transcript+variants+by+subtractive+transcriptome-wide+analysis.">A global view of cancer-specific transcript variants by subtractive transcriptome-wide analysis.</a> PloS one. 4 (3), 4732 (2009).</li><li>Venables, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+alternative+splicing+markers+for+breast+cancer.">Identification of alternative splicing markers for breast cancer.</a> Cancer Res. 68 (22), 9525-9531 (2008).</li><li>Shapiro, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+EMT-Driven+Alternative+Splicing+Program+Occurs+in+Human+Breast+Cancer.">An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer.</a> PLoS Genet. 7 (8), 1002218 (2011).</li><li>David, C. J., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+pre-mRNA+splicing+regulation+in+cancer:+pathways+and+programs+unhinged.">Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged.</a> Genes Dev. 24 (21), 2343-2364 (2010).</li><li>Ladomery, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+alternative+splicing+is+another+hallmark+of+cancer.">Aberrant alternative splicing is another hallmark of cancer.</a> Int J Cell biol. 2013, (2013).</li><li>Karni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gene+encoding+the+splicing+factor+SF2/ASF+is+a+proto-oncogene.">The gene encoding the splicing factor SF2/ASF is a proto-oncogene.</a> Nat Struct Mol Biol. 14 (3), 185-193 (2007).</li><li>Anczuk&#242;w, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRSF1-regulated+alternative+splicing+in+breast+cancer.">SRSF1-regulated alternative splicing in breast cancer.</a> Mol cell. 60 (1), 105-117 (2015).</li></ol>

作者没有什么可以披露的。

本报告详细描述了可以特异性操纵靶基因选择性剪接的人工剪接因子的设计和构建。该方法利用PUF重复的独特的RNA结合模式产生具有定制特异性的RNA结合支架。它可以用于激活或抑制拼接。 该协议中的关键步骤是生成定义了ESF特异性的重新编程的PUF结构域。已经开发并优化了PCR拼接方案，用于快速生成PUF支架。其成功的关键是将不同重叠模板的比例调整为1:1:1。每次循环后PCR产物的纯化也至关重要，因为未纯化的产品可能会含有上一轮的底漆污染。另一个重要的步骤是使用半定量RT-PCR来测定剪接比率。一般也是男人ý扩增循环应该避免，因为它们可能饱和PCR反应。在我们与剪接记者的共表达的实验中，20 - 25个循环进行了常规使用的，但是这可以根据丰度mRNA的测量内源基因的剪接时变化。当使用新的引物对量化剪接异构体，建议校准每次PCR的实验中，如先前所描述16。 与设计师ESFS的潜在限制在于它们的脱靶效应，因为该特异性是由在PUF支架重复的次数来确定。野生型PUF识别的8个核苷酸位点，这与通过识别其靶的siRNA的特异性"种子匹配"。然而，由于任意的8个核苷酸序列可能偶然在转录65000出现一次NT长（4 8 = 65536），将有由设计者PUF认可的其他脱靶转录物。脱离目标的EFF可以通过使用与另外的重复的PUF减小ECT;然而，它仍然是评估的特异性和ESFS的脱靶效应非常有用。以最小化潜在的脱靶效应，在较低的水平多设计师ESFS的组合的表达也可以进行。在这种情况下，关断目标基因可以不被ESFS的低量的影响，而真正的目标的剪接将由协同起作用的多个ESFS受到影响。该解决方案是类似于在基因沉默使用的RNAi，其中靶向单个mRNA的多个位点的汇集的siRNA（各以降低的浓度）可降低脱靶效应的研究人员。 其他主要方法来操纵AS是使用反义寡核苷酸与所述mRNA前体的某些区域配对。与此相比现有的方法，该ESFS可导致在稳定转染的细胞延长的效果。此外，E的体内递送SF可以利用增加的基因治疗载体的武库，而反义寡核苷酸的体内递送非常难以控制。此外，该方法可以避免寡核苷酸的复杂且昂贵的修饰。使用各种诱导型启动子，也可以在正确的细胞类型和正确的时间更精确地控制ESF表达。该方法的主要缺点是相对较低的特异性（8-nt识别位点与典型反义寡核苷酸中的16至20-nt识别位点）。 选择性剪接的失调导致许多疾病，包括癌症26,27 。全基因组研究已经揭示了各种类型的癌症28,29,30 中超过15,000个肿瘤相关的剪接变体。例如，intronic肿瘤抑制基因的剪接位点突变通常导致外显子跳跃事件，并产生可能有助于肿瘤发生的异常蛋白质26,31,32。此外，发现一些剪接因子在许多癌症类型中过表达，这有助于细胞转化33,34 ，表明剪接因子也可以在癌症生物发生中起重要作用。因此，除了提供调节基因功能的有用工具之外，用设计者ESF进行拼接的操作还可能恢复癌症中的错配剪接事件，从而提供潜在的治疗工具。此外，通过将设计者PUF结构域与不同功能域融合，可以设计操纵各种RNA代谢过程的多种人为因子。例如，翻译激活物（GLD2）或翻译代谢物的融合与PUF结构域的融合蛋白（CAF1）产生可激活或抑制mRNA翻译的新因子27 。使用相同的设计原则，我们将非特异性RNA核酸内切酶结构域（PIN）与一系列设计者PUF结构域结合，以产生类似于DNA限制酶17的功能的人工位点特异性RNA内切核酸酶（ASRE）。

大多数的人类基因经历选择性剪接（AS），以产生多种同种型具有不同的活动，这大大增加了基因组1,2的编码的复杂性。 AS提供的主要机制来调节基因的功能，并且在不同的细胞和发育阶段3,4通过各种途径紧密调节。因为拼接错误调节是人类疾病5，6，7，8的常见原因，靶向剪接调节正在成为一种有吸引力的治疗路线。 根据剪接调节的简化模型，如由剪接调节顺 -elements（SRES）中前mRNA剪接增强子或替代外显子沉默发挥功能的主要控制。钍ESE的SRE具体招募各种反式作用蛋白因子促进或抑制剪接反应3,9（ 即剪接因子）。大多数反式作用剪接因子有单独的序列特异性RNA结合域认识到自己的目标和效应域控制剪接。最熟知的例子是丝氨酸/富含精氨酸的（SR）蛋白家族的含有N-末端的RNA识别基序（RRMS），其结合外显子剪接增强子，和C-末端RS结构域的成员，其中促进外显子内含10 。相反地，核蛋白A1通过RRM结构域结合至外显子剪接沉默子，并通过C末端的富含甘氨酸的结构域11抑制外显子包含。使用这种模块化配置，研究人员应该能够通过合并来设计人工剪接因子特异性RNA结合结构域（RBD）具有不同的短跑运动员激活或抑制剪接的结构域。 这种设计的关键是使用可编程RNA结合特异性识别给定靶标的RBD，其类似于TALE结构域的DNA结合模式。然而，大多数天然剪接因子含有RRM或K同源性（KH）结构域，其识别具有弱亲和力的短RNA元件，因此缺乏预测性RNA-蛋白质识别"代码" 12 。 PUF重复蛋白（ 即 PUF结构域）的RBD具有独特的RNA识别模式，允许重新设计PUF结构域以特异性识别不同的RNA靶标13,14 。典型的PUF结构域包含八个重复的三个α螺旋，每个重复在8-nt RNA靶标中识别单个碱基。第二个α-螺旋的某些位置的氨基酸侧链与t的Watson-Crick边缘形成特定的氢键他的RNA碱基决定了每个重复的RNA结合特异性（ 图1A ）。 PUF重复的RNA碱基识别代码令人惊讶地简单（ 图1A ），允许产生识别任何可能的8碱基组合的PUF结构域（由Wei和Wang 15综述）。 该模块化设计原则允许产生由定制的PUF域和剪接调制域（ 即 SR域或富含Gly域）组成的工程化拼接因子（ESF）。这些ESFs可以作为剪接活化剂或作为抑制剂来控制各种类型的剪接事件，并且已被证明可用作操纵与人类疾病相关的内源基因的剪接16,17 。例如，我们已经构建了PUF-Gly型ESFs来特异性地改变Bcl-x基因的剪接，将抗凋亡长的同种型（Bcl-xL）转化为促细胞凋亡的短异构体（Bcl-xS）。改变Bcl-x同种型的比例足以使几种癌细胞对多种抗癌化学药物16敏感，这表明这些人为因子可能作为潜在的治疗试剂。 除了用已知的剪接效应子结构域（ 例如， RS或富含Gly的结构域）控制剪接之外，工程化PUF因子也可用于检查新的剪接因子的活性。例如，使用这种方法，我们已经证明，当结合到不同的前mRNA区域18时，几种SR蛋白质的C-末端结构域可以激活或抑制剪接，RBM4的富含丙氨酸的基序可以抑制剪接19 ，并且DAZAP1的富含脯氨酸的基序可以增强剪接20,21/ sup&gt;。这些新功能域可用于构建其他类型的人为因子以微调拼接。

<table><tbody><tr><td>High-fidelity DNA polymerase (Phusion High-Fidelity) with PCR buffer</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>DNA ligase (T4 DNA ligase)</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>Liposomal transfection reagent (Lipofectamine 2000)</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Reduced serum medium (Opti-MEM)</td><td>Gibco</td><td>31985-062</td><td></td></tr><tr><td>RNA extraction buffer (TRIzol Reagent)</td><td>ambion</td><td>15596018</td><td>TRIzol reagent includes phenol, which can cause burns. Wear gloves when handling</td></tr><tr><td>PBS (1x)</td><td>Life Technologies</td><td>10010-031</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase (RT)</td><td>Invitrogen</td><td>18080044</td><td></td></tr><tr><td>Caspase-3 antibody</td><td>Cell Signaling Technology</td><td>9668</td><td></td></tr><tr><td>PARP antibody</td><td>Cell Signaling Technology</td><td>9542</td><td></td></tr><tr><td>Bcl-x antibody</td><td>BD Bioscience</td><td>610211</td><td></td></tr><tr><td>beta-actin antibody</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>alpha-tubulin antibody</td><td>Sigma-Aldrich</td><td>T5168</td><td></td></tr><tr><td>FLAG antibody</td><td>Sigma-Aldrich</td><td>F4042</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Amersham-Pharmacia</td><td>RPN203D</td><td></td></tr><tr><td>ECL Western Blotting detection reagents</td><td>Invitrogen</td><td>WP20005</td><td></td></tr><tr><td>Cy5-dCTP</td><td>GE Healthcare</td><td>PA55021</td><td></td></tr><tr><td>Fluorescence-activated Cell Sorter (FACS)</td><td>BD Bioscience</td><td>FACSCalibur&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma</td><td>P4170</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7638-5G</td><td></td></tr><tr><td>Triton-X100</td><td>Promega</td><td>H5142</td><td></td></tr><tr><td>Poly-L-Lysine&amp;nbsp;</td><td>Sigma</td><td>P-4832</td><td>Filter-sterilize and store at 4 &amp;deg;C</td></tr><tr><td>Vector pWPXLd</td><td>Addgene</td><td>12258</td><td></td></tr><tr><td>Vector pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>Vector psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>DNase I (RNase-free)</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>Oligo(dT)&lt;sub&gt;18&lt;/sub&gt; Primer</td><td>Thermo Scientific</td><td>SO131&amp;nbsp;</td><td></td></tr><tr><td>Anti-mouse secondary antibody (Anti-mouse IgG, HRP-linked Antibody)</td><td>Cell Signaling Technology</td><td>7076S</td><td></td></tr></tbody></table>

engineering artificial factors to specifically manipulate alternative splicing in human cells

大多数真核RNA的处理是由RNA介导的结合的模块化配置，包括一个RNA识别模块，其特异性地结合前mRNA靶和效应结构域的蛋白质（限制性商业惯例）。以前，我们已经利用所述PUF结构域的独特RNA结合模式在人类Pumilio 1，以产生一个可编程的RNA结合的支架，将其用于设计各种人造限制性商业惯例来操纵RNA代谢。这里，详细的协议描述来构建专门设计用于调节靶基因的可变剪接工程剪接因子（ESFS）。该协议包括如何设计和构建定制的PUF支架用于特定RNA靶，如何通过融合设计者PUF结构域和效应子结构域，以及如何使用ESFS来操纵目标基因的剪接以构建ESF表达质粒。在该方法的代表性结果，我们还描述了常见的测定法的使用拼接记者，ESF的培养的人细胞中的应用，和剪接改变后续效应ESF活动。按照本报告中的详细协议，它可以设计并为不同类型的选择性剪接（AS）的规ESFS，提供了一个新的战略研究剪接调节和不同的剪接异构体的功能。此外，通过用设计PUF域融合不同的功能结构域，研究人员可以设计靶向特定RNA操纵RNA加工的各种步骤人为因素。

这份报告描述了ESFS和拼接记者的设计和施工的完整的协议。它还概述ESFS的操纵内源基因16的AS的进一步应用。为了说明的ESF介导的拼接变化的典型结果，我们使用的数据来自我们以前的工作作为一个例子。具有不同的功能结构域的ESFS可用于促进或抑制靶盒外显子（ 图1 D＆E）的夹杂物。 ESFS也可影响在报道系统（ 图1 F＆G）替代5&#39;和3&#39;剪接位点的使用。 内源基因的可变剪接也可以与设计师ESFS进行具体调整。我们已经证明了通过规范这个应用程序ifically靶向的Bcl-x，它可以被拼接成具有替代5&#39; 剪接位点的两种拮抗同种型。我们设计了一个ESF，甘氨酸 - PUF（531），识别备选5&#39; 剪接位点之间的8个核苷酸的RNA元件。此甘氨酸- PUF（531）具体地移位剪接朝向生产的Bcl-XS（ 图2 A）的。转染甘氨酸 - PUF（531）到HeLa细胞和Bcl-XS亚型和Bcl-XS蛋白以剂量依赖性方式增加的电平，而控制ESF后，甘氨酸 - PUF（WT），没有影响的比率的Bcl-XS的到Bcl-xL的（ 图2 B和C）。此外，设计者可以ESF诱导胱天蛋白酶3和聚（ADP-核糖）聚合酶（PARP），两个公知的细胞凋亡的分子标志物（ 图2 D）的切割。如所预期的，设计者ESFS被主要定位在转染细胞的核，作为演示通过免疫荧光显微镜nstrated（ 图2 E）。一致的是，通过甘氨酸- PUF剪接移（531）引起的核DNA的碎裂，这表明这些细胞经历凋亡（ 图2 E）。凋亡细胞的增加通过检查从随机选择的视野多于200个细胞并通过量化具有片段化DNA的核（ 图2 F）的细胞的百分比进一步证实。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/54967/54967fig1.jpg" /> 图1: 设计ESFS和它们在调节外显子跳跃活动。 （A）特定的PUF结构域和RNA靶标之间的结合被示出与RNA-PUF结构和示意图。为每个所述PUF绑定代码4个RNA碱基，与不同颜色的右侧所示，用于设计PUF突变。 （B）的流程图，以获得一个定制的PUF结构域。识别"UGUAUAUA"的PUF用作一个例子。 A 4轮PCR策略是用于组装定制RNA结合特异性（颜色类似编码到面板A）一个PUF支架。在第一轮中，并入两个相邻PUF重复所需的RNA识别码的一系列PCR引物被用于产生四个片段包括一个完整的PUF蛋白质的八个RNA识别码（R1 / R2，R3 / R4 ，R5 / R6和R7 / R8）。编码N末端核定位信号，一个C-末端终止密码子帽片段和桥片段也单独制造（5&#39;-端和3&#39;-端帽，桥2/3，桥4/5，和桥6 / 7）。在第二轮中，通过混合编码与适当的桥相邻重复的重叠片段（ 例如，混合R1 / R2生成的新模板，R3 / R4和桥2/3生成R1 - 4的模板），然后用DNA聚合酶扩展填补空白。类似地，第三轮连接R1-4，R5-8和桥4/5。最后，第四轮将PUF结构域的5&#39;末端和3&#39;末端连同克隆位点一起加入到表达载体中。 （ C ）ESF的模块化域名组织。 ESF由CMV启动子（箭头）驱动，并从N至C末端编码:FLAG表位（用于检测ESF），功能模块（富含Gly的结构域或RS结构域），NLS （促进ESF的核定位）和RNA识别结构域（PUF结构域）。 Nhe I和BamH I被设计为插入功能模块，而Xba I和Sal I被设计为插入RNA识别结构域。 （ D ）Gly-PUF ESF与外显子跳过报告共同表达，通过RT-PCR分析剪接模式。改性PUF a </s向上&gt;和PUF b特异性结合8-mer的目标A和B，分别为（在相同的颜色）。所有组合被使用，因此不同颜色的PUF目标对作为对照。 RS-PUF的上外显子跳跃（E）的影响，竞争5&#39;剪接位点（F），或竞争性3&#39;剪接位点记者（G）通过类似于面板D.方法RT-PCR的数据测定从Wang 等人。 16。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54967/54967fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/54967/54967fig2.jpg" /> 图2: 与内源性ESFS的Bcl-X前mRNA剪接的调节。 （A）的内源性BC的可变剪接的示意LX mRNA前体。在的Bcl-x的外显子2的两个替代5&#39; 剪接位点被用来生成不同尺寸的两种同种型，Bcl-xL的和Bcl-XS。两个5&#39; 剪接位点之间的序列UGUGCGUG被选择作为目标ESF和WT PUF重复1，图3和5（Q867E / Q939E / C935S / Q1011E / C1007S）重新编程（星号）来识别该靶序列。含有丰富的甘氨酸域所得ESF禁止将下游5&#39; SS（红色箭头指示）。的Bcl-X 5&#39; SS使用的（B）调制。不同量的甘氨酸 - PUF（531）表达构建体转染到HeLa细胞中。甘氨酸 - PUF（WT）被用作对照。使用对应于外显子1和的Bcl-X基因的3个引物通过RT-PCR检测到的Bcl-x的两种同种型。所述的Bcl-XS同种型的百分比进行定量，并在底部示出。 （C）ESFS影响了Bcl-xL和Bcl-XS的表达水平。将样品以相同的顺序加载在面板B中，和所有蛋白质ARe被Western印迹检测。通过抗FLAG抗体检测ESF的表达，将微管蛋白水平用作对照。 （ D ）将不同量的ESF表达构建体转染到HeLa细胞中，导致PARP和半胱天冬酶3的切割。转染24小时后通过Western印迹检测样品。肌动蛋白水平被检测为对照。 （ E ）通过免疫荧光显微镜检测转染HeLa细胞中ESFs的亚细胞定位。细胞与DAPI共染色显示细胞核。一些核，特别是在用Gly-PUF（531）转染的细胞中，由于细胞凋亡而被分裂。刻度棒:5μm。 （ F ）从随机选择的荧光显微镜图像领域测量凋亡细胞（ 即具有片段核DNA的细胞）的百分比。条形表示平均值，而点表示来自两个实验的数据。这个数字s是我们早些时候的报告由Wang 等人修改。 16，根据自然出版集团的政策。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54967/54967fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

该报告介绍了一种生物工程方法来设计和构建特异性调节在哺乳动物细胞中靶基因的剪接新颖人工剪接因子（ASFS）。该方法可以进一步扩展来设计各种人为因素来操纵RNA代谢的其它方面。

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

engineering-artificial-factors-to-specifically-manipulate-alternative

Nanyang Technological University

Research

JoVE Journal

Genetics

8.9K 次观看.  Shanghai Institutes for Biological Sciences (SIBS). 该报告介绍了一种生物工程方法来设计和构建特异性调节在哺乳动物细胞中靶基因的剪接新颖人工剪接因子（ASFS）。该方法可以进一步扩展来设计各种人为因素来操纵RNA代谢的其它方面。 

视频: Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

科研

癌症研究

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

发育生物学

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

遗传学

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

生物化学

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

使用 E1A 微型工具研究 mRNA 拼接更改

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

生物学

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

RNA-seq数据中替代剪接和聚腺苷酸化的鉴定

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

RNA 剪接

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...