Name: describing a transcription factor dependent regulation of the microrna transcriptome
Uploaded: 2016-06-15T13:00:00.000+00:00
Duration: 7 min 23 s
Description: Watch this Scientific Journal Video about Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome at JoVE.com

这项研究是由来自全球CLL研究基金会的资助。得克萨斯大学MD安德森癌症中心的大学是由美国国立卫生研究院的部分通过一个癌症中心支援津贴（P30CA16672）的支持。

1.识别微小RNA基因的启动子转录因子结合位使用数据挖掘方法<ol><li>使用美国加州大学圣克鲁兹分校（UCSC）基因组浏览器作为提取DNA元素（ENCODE）项目的百科全书的一部分生成染色质免疫沉淀（芯片）测序数据。 <ol><li>打开在UCSC基因组浏览器表浏览器。 </li><li>使用以下规格提取表:类群:（哺乳动物），基因组（人），大会:（Feb2009（GRch37 / hg18）），组（监管），跟踪（TxnFactorChIP），表（weEncoderesTFbsCo7steredv3），区域（基因组），输出格式（从选定表中的所有基因）。 </li><li>从保存1.1.2输出为.txt文件，并到电子表格中。 </li><li>排序和过滤感兴趣的转录因子（ 例如 ，STAT2）。 </li></ol></li><li>使用基于H3K4me3的表观遗传签名的microRNA发起人提供Baeer 等人名单。9 </suP&gt;。这个列表复制到一个文本文件。 </li><li>根据人类基因组坐标相匹配时，数据从1.1和1.2（ 例如 ，STAT3上推定的微小RNA的启动子结合）映射，并使用C语言编写的尖锐的代码确定为在补充所述编码文件1中位结合亲和力。 </li></ol> 2.使用的shRNA下调利率的转录因子的表达<ol><li>板从于10cm板293人胚肾细胞系在约50％汇合（含10％FBS）的1.5×10 6个细胞。 </li><li>从用含有shRNA的绿色荧光蛋白（GFP）的慢病毒的5微克293人胚肾细胞系转染细胞定向到感兴趣的转录因子，并与使用转染试剂，根据制造商的协议的贴壁细胞的包装载体的5微克。 </li><li>作为对照，转染从293人胚肾细胞系的细胞加扰的shRNA和根据制造商的协议的包装载体。 </li><li>保持对细胞转染混合物16小时（在37℃，CO 2培养箱），然后改变媒体至10ml新鲜的10％DMEM培养基含10％FBS。 </li><li>等待48小时转染后，离心细胞培养物（300 XG，5分钟），并收集感染上清。通过0.45μm注射器式滤器（25毫米无表面活性剂的醋酸纤维素膜）过滤上清液以除去任何漂浮的细胞。 </li><li>浓缩上清液，用超离心过滤装置与100 kDa的门槛收集慢病毒。旋转950 XG 30 - 60分钟，直到量一直精矿小于250微升。在-80℃保存浓缩病毒。 </li><li>转染用慢病毒的细胞。从-80℃冷冻机中取出冷冻的慢病毒和解冻至室温。转移100μl病毒上清到一个新的1.5 ml离心管。 <ol><li>布林克音量在管到1ml具有降低的血清培养基。聚凝胺添加到1ml病毒悬浮液为10ng / ml的终浓度，轻轻混合并让该混合物静置5分钟。 </li></ol></li><li>离心机5×10 6个细胞的每个转导并轻轻重悬细胞沉淀在0.5ml的含有病毒的介质。让细胞留在培养箱中4 - 24小时，然后加入0.5毫升20％FBS的培养基中，以10％的FBS的最终浓度。 </li><li>等待48至72小时，并弄脏用碘化丙锭（PI），并根据制造商的说明绿色荧光蛋白（GFP）的细胞。避光细胞，并用流式细胞分选仪来测量GFP + / PI的价格-细胞。由于PI污渍只有死细胞，这一比率在活细胞的转染效率的估计。 </li><li>如前面所述10排序的阳性细胞数来GFP表达（GFP +）通过FACS分拣机。 </li><li>利用Weste氡免疫印迹如前所述10之前，并与指定的shRNA感染感兴趣的细胞（例如，白血病细胞）后，以确定所关注的转录因子的水平。 </li></ol> 3.确定微RNA转录的表达水平与转录因子的shRNA转染细胞<ol><li>使用商业试剂盒根据制造商的协议的RNA分离。 </li><li>标记的RNA和其杂交到微RNA微阵列11。 </li><li>确定与转录因子的shRNA或空载体对照5转染的细胞中差异表达的microRNA。 </li><li>验证用于使用实时PCR 5的最差异表达的微小RNA的微阵列结果。 </li></ol> 4.确定生物信息学和shRNA的途径之间的重叠在描述转录因子相关转录<ol><li>要DET貂窝藏转录因子结合在其发起人地点和转录因子的shRNA表达下调转染细胞的microRNA基因的预期和观察到的比例，做到以下几点: <ol><li>获得窝藏感兴趣的转录因子在从1.3生成的列表中的启动子/总微小RNA基因的比率。这个名单是预期的比率（ 例如 ，微小RNA基因用STAT3结合位点/总微小RNA基因测试= 0.25）。 </li><li>确定在转录因子的shRNA转染的细胞下调从3.3生成的列表并且具有从1.3生成的列表兴趣结合位点的转录因子的基因的数目。下调的基因的这个数/总数是所观察到的比例（ 例如 ，微小RNA基因用STAT3结合位点/总数下调微小RNA = 0.6）。 </li></ol></LI&gt; <li>使用χ2统计比较上述生成的观察值与预期的比率，并确定是否在转录因子的shRNA转染的细胞下调的基因的列表与在其启动子的转录因子结合位点富集。 </li></ol>

<ol>
	<li>Bentwich, 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=Identification+of+hundreds+of+conserved+and+nonconserved+human+microRNAs.">Identification of hundreds of conserved and nonconserved human microRNAs.</a> Nat Genet. 37, 766-770 (2005).</li><li>Hata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+microRNAs+in+cardiovascular+biology+and+disease.">Functions of microRNAs in cardiovascular biology and disease.</a> Annu Rev Physiol. 75, 69-93 (2013).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Piriyapongsa, J., Jordan, I. K., Conley, A. B., Ronan, T., Smalheiser, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+sites+are+highly+enriched+within+microRNA+precursor+sequences.">Transcription factor binding sites are highly enriched within microRNA precursor sequences.</a> Biol Direct. 6, 61 (2011).</li><li>Rozovski, U., 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=Signal+transducer+and+activator+of+transcription+(STAT)-3+regulates+microRNA+gene+expression+in+chronic+lymphocytic+leukemia+cells.">Signal transducer and activator of transcription (STAT)-3 regulates microRNA gene expression in chronic lymphocytic leukemia cells.</a> Mol Cancer. 12, 50 (2013).</li><li>Turner, M. J., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+microRNA+expression+in+C.+elegans:+promoting+better+understanding.">Transcriptional control of microRNA expression in C. elegans: promoting better understanding.</a> RNA Biol. 6, 49-53 (2009).</li><li>Cui, Q., Yu, Z., Pan, Y., Purisima, E. O., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+preferentially+target+the+genes+with+high+transcriptional+regulation+complexity.">MicroRNAs preferentially target the genes with high transcriptional regulation complexity.</a> Biochem Biophys Res Commun. 352, 733-738 (2007).</li><li>Yamakuchi, M., Lowenstein, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-34,+SIRT1+and+p53:+the+feedback+loop.">MiR-34, SIRT1 and p53: the feedback loop.</a> Cell Cycle. 8, 712-715 (2009).</li><li>Baer, C., 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=Extensive+promoter+DNA+hypermethylation+and+hypomethylation+is+associated+with+aberrant+microRNA+expression+in+chronic+lymphocytic+leukemia.">Extensive promoter DNA hypermethylation and hypomethylation is associated with aberrant microRNA expression in chronic lymphocytic leukemia.</a> Cancer Res. 72, 3775-3785 (2012).</li><li>Hazan-Halevy, 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=STAT3+is+constitutively+phosphorylated+on+serine+727+residues,+binds+DNA,+and+activates+transcription+in+CLL+cells.">STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells.</a> Blood. 115, 2852-2863 (2010).</li><li>Melo, S. A., 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+TARBP2+mutation+in+human+cancer+impairs+microRNA+processing+and+DICER1+function.">A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function.</a> Nat Genet. 41, 365-370 (2009).</li><li>Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+STAT+family+proteins:+lessons+from+knockout+mice.">Functional roles of STAT family proteins: lessons from knockout mice.</a> Stem Cells. 17, 138-146 (1999).</li><li>Frank, D. A., Mahajan, S., Ritz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+lymphocytes+from+patients+with+chronic+lymphocytic+leukemia+contain+signal+transducer+and+activator+of+transcription+(STAT)+1+and+STAT3+constitutively+phosphorylated+on+serine+residues.">B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues.</a> J Clin Invest. 100, 3140-3148 (1997).</li><li>Calin, G. A., 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=MicroRNA+profiling+reveals+distinct+signatures+in+B+cell+chronic+lymphocytic+leukemias.">MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias.</a> Proc Natl Acad Sci U S A. 101, 11755-11760 (2004).</li><li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+the+encyclopedia+of+DNA+elements+(ENCODE).">A user's guide to the encyclopedia of DNA elements (ENCODE).</a> PLoS biology. 9, e1001046 (2011).</li><li>Miranda, K. C., 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+pattern-based+method+for+the+identification+of+MicroRNA+binding+sites+and+their+corresponding+heteroduplexes.">A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes.</a> Cell. 126, 1203-1217 (2006).</li><li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489, 57-74 (2012).</li><li>Lau, J. C., Hanel, M. L., Wevrick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+and+imprinted+epigenetic+modifications+of+the+human+NDN+gene.">Tissue-specific and imprinted epigenetic modifications of the human NDN gene.</a> Nucl Acids Res. 32, 3376-3382 (2004).</li><li>Corcoran, D. L., 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=Features+of+mammalian+microRNA+promoters+emerge+from+polymerase+II+chromatin+immunoprecipitation+data.">Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data.</a> PLoS One. 4, e5279 (2009).</li><li>Bhattacharyya, M., Feuerbach, L., Bhadra, T., Lengauer, T., Bandyopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+transcription+start+site+prediction+with+multi-objective+feature+selection.">MicroRNA transcription start site prediction with multi-objective feature selection.</a> Stat Appl Genet Mol Biol. 11, (2012).</li></ol>

The authors declare no competing financial interests.

蛋白质编码基因的RNA聚合酶II-依赖性转录背后的机制已被广泛研究。而这些元素组成只有1％ -人类基因组的2％，从ENCODE项目证据表明，人类基因组的80％以上的可经历转录17和什么调节非编码DNA元件的转录在很大程度上仍然未知6 。 几项研究，表明聚合酶II也负责的一些非编码蛋 ​​白的基因，包括微RNA 6的转录，导致我们开发出从公共可用的资源，?...

微RNA是内生的小非编码调控RNA，通常在转录后水平起作用的mRNA表达的作为负调节。大约1000非编码20至25个核苷酸长的微小RNA在人类基因组中的1,2-找到。微RNA调节通过微RNA及其互补种子匹配序列，其通常位于3&#39;非翻译区的靶mRNA的（UTR）的种子序列之间规范碱基配对的基因表达。总的来说，微RNA调控的蛋白质编码基因3超过30％，但仅知之甚少从微RNA的DNA的转录。它已被提出，微RNA转录的调节类似于表达4,5。特别是，类似于其在促进蛋白质编码基因的转录活性，转录因子被认为激活微RNA 6的转录。转录因子-MicroRNA相互作用已被报告为基因表达7的调制因子，并且还可以形成反馈和前馈回路。例如，Yamakuchi 等报道的反馈环路，其中的p53诱导microRNA34a，表达这反过来又抑制了p53的阻遏SIRT的翻译，并由此提高p53活性8。 而微RNA的转录因子依赖性表达的具体例子，已报道，缺乏其中提供了关于感兴趣的转录因子如何调节微小RNA-转录的表达信息的被接受的方法。本文所提出的协议的目的是提供微小RNA-转录的转录依赖性因子调控的进行了深入的说明。通过结合公开可用的数据，生物信息学工具，并使用微阵列技术，谁遵循这个算法的研究人员将能够捕获基因组规模如何的任何在任何感兴趣的细胞类型的转录因子调节微小RNA-转录的表达和在调节微RNA表达探索转录因子mRNA的推定贡献。

<table><tbody><tr><td>Lipofectamin 2000</td><td>Life Technologies</td><td>11668027</td><td></td></tr><tr><td>0.45 &amp;micro;m syringe filter</td><td>Thermo Scientific (Nalgene)</td><td>190-2545</td><td></td></tr><tr><td>Amicon ultracentrifugal filter device with threshold of 100&amp;nbsp;kDa</td><td>Merck Millipore</td><td></td><td></td></tr><tr><td>Polybrene</td><td>Merck Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td>TRIzol reagent</td><td>Life Tachnologies (Invitrogen)</td><td>15596-026</td><td></td></tr><tr><td>293 Cell line human</td><td>Sigma-Aldrich</td><td>85120602</td><td></td></tr></tbody></table>

describing a transcription factor dependent regulation of the microrna transcriptome

而蛋白质编码基因的转录调控被广泛研究，知之甚少转录因子如何参与非编码RNA的转录，特别的微RNA。在这里，我们提出了一个战略，研究使用规范公开提供的数据，计算资源和高吞吐量数据的microRNA的转录转录因子的潜在作用。我们使用的H3K4me3的后生签名从ENCODE项目识别微RNA启动子和染色质免疫沉淀（ChIP）-sequencing数据，以确定被富含转录因子结合位点的微RNA启动子。通过转染与shRNA的靶向感兴趣的转录因子和使细胞微RNA阵列感兴趣的细胞中，我们研究这转录因子对微RNA转录的影响。作为一个说明性的例子，我们使用我们的研究STAT3的慢性Ly的微小RNA转录的影响mphocytic性白血病（CLL）细胞。

STAT3是其通常导致具有抗凋亡和增殖作用12个基因的转录的转录因子。无论是STAT3也影响了非编码RNA的转录，目前尚不得而知。在所有CLL细胞STAT3是丝氨酸残基707组成10,13磷酸化。磷酸STAT3梭到细胞核，与DNA结合，并激活已知由酪氨酸pSTAT3的在其他细胞类型10激活的基因。因为CLL特征是微小RNA网络14的全球解除管制，我们推测，丝氨酸pSTAT3的的...

Watch this Scientific Journal Video about Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome at JoVE.com

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

描述了微RNA转录的转录因子依赖调节

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

describing-transcription-factor-dependent-regulation-microrna

Research

JoVE Journal

Bioengineering

8.3K 次观看.  Rabin Medical Center. Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest. 

视频: Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

科研

遗传学

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

癌症研究

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

免疫与感染

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

生物学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

The Ago2-miRNA-co-IP Assay to Study TGF- &#946;1  Mediated Recruitment of miRNA to the RISC in CFBE Cells

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-&#946;1 (TGF-&#946;1). However, little is known about whether the TGF-&#946;1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-&#946;1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-&#946;1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Here, we describe the Ago2-miRNA-co-IP assay designed to quantify an active pool of specific miRNAs induced by TGF-&#946;1 in human bronchial epithelial CFBE41o- cells. This assay provides functional information on the recruitment of miRNA to the RNA induced silencing complex, quantified by qRT-PCR using specific miRNA primers and TaqMan hydrolysis probes.

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

用于微RNA簇网络分析的CRISPR基因编辑工具

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

Regulation of Expression Occurs at Multiple Steps

Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the binding of proteins to short regulatory sequences on the DNA. This association can either promote or inhibit the transcription of a gene associated with the respective sequence.
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a protein. This happens through mRNA splicing, which involves the removal of non-coding regions and merging of the coding ones. mRNA processing can also be used as a regulatory mechanism through variation in splicing patterns such as skipping of certain exons, alternative splicing, and inclusion of introns.&#160;
The addition of a poly-A tail at the 3&#8217; end and a 5&#8217; cap to produce the mature mRNA are also regulatory points during RNA processing. Regulation occurs through variation in the polyadenylation signal, which determines where the poly-A tail will be added onto the mRNA.&#160; In some cases, more than one poly-A signal is present at the 3&#8217; end which will change the length of the 3&#8217; untranslated region, but the final protein product will be the same. However, the stability and the translation potential the variants mRNA may differ, which can alter the amount of protein produced. In other cases, an additional poly-A signal is present on the intron or exon within the gene sequence which may lead to variation in splicing sites for polyadenylation and result in different proteins from the same strand of pre-mRNA.The addition of the 5&#8217; cap, which is made up of methylated guanosine, is regulated by two mechanisms.&#160; One involves the regulation of the methyltransferases that add the methyl group to the guanosine, and the other is through regulation of the cellular signaling pathways that lead to methylation.
Next, the mature mRNA needs to be transported from the nucleus to the cytoplasm through nuclear pore complexes (NPCs) to be translated. This is regulated by the mRNA to forming a complex, known as the ribonucleoparticle, with RNA binding proteins. NPCs only allow mRNAs that are in the complex to pass into the cytoplasm. Once an mRNA enters the cytoplasm for translation, it can either be targetted individually or as a part of a group through specific regulations, or it can undergo a common regulation with all other mRNAs in the cytoplasm. In specific regulation, particular trans-acting elements, like proteins and different types of RNAs, regulate transcription. In general regulation, the proteins involved in the translation machinery are activated or inhibited, which in turn affects the translation of all transcripts. The most common translational regulatory mechanism is the modification of the translation initiation factor.
Gene expression can also be regulated through post-translational modifications, where an enzyme-catalyzed reversible modification can alter the function of a protein. A common post-translational modification is phosphorylation, which is carried out by enzymes known as kinases. Dephosphorylation of proteins, on the other hand, is carried out by proteins known as phosphatases. Phosphorylation of a protein can result in its activation or deactivation and alter its function.

Regulation of Expression during Transcription and Translation

表达调节发生在多个步骤

Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the ...

教育

JoVE Core

分子生物学

Unit 1

基因表达

关键术语与概念

Regulation of Expression at Multiple Steps

The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors&#8212;these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5&#39; capping, splicing, and the addition of a 3&#39; poly-A tail, forming the mature mRNA. Only those mature mRNAs that associate with RNA-binding proteins (RBPs) forming ribonucleoprotein particles are prevented from degradation and are selectively transported out of the nucleus for protein synthesis. mRNAs that do not associate with RBPs are not transported out of the nucleus and hence not translated into proteins. Alternatively, if a mature mRNA binds a complementary microRNA, it undergoes degradation, thereby inhibiting protein synthesis.
However, once the mRNA is translated, the newly synthesized protein undergoes modification that affects its activity. For example, adding functional groups, such as methyl groups, phosphate, or acetyl groups, may activate or inactivate the protein. In contrast, adding multiple ubiquitin proteins to a substrate protein marks them for degradation. Thus, ubiquitination regulates protein stability and its functional activity.

多步骤表达调节

The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. ...

描述了微RNA转录的转录因子依赖调节

摘要

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