Name: par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins
Uploaded: 2010-07-02T17:00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

نشكر أعضاء Tuschl المختبر لإجراء مناقشات مفيدة. معتمد من قبل الشقيقين MH Akademischer الألماني (DAAD). وأيد هذا العمل من قبل الصندوق الوطني السويسري المنحة # 3100A0 - 114001 لMZ ؛ TT غير محقق HHMI ، وكان يؤيد العمل في مختبره من المنح والمعاهد الوطنية للصحة GM073047 MH08442 ومؤسسة ستار.

<ol>
	<li>Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat. Rev. Genet. 8 (7), 533-533 (2007).</li><li>Tenenbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc. Nat. Acad. Sci. 97 (26), 14085-14085 (2000).</li><li>Gerber, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+mRNAs+associated+with+the+translational+regulator+PUMILIO+in+Drosophila+melanogaster.">Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster.</a> Proc. Nat. Acad. Sci. 103 (12), 4487-4487 (2006).</li><li>Lopez de Silanes, I. <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+a+target+RNA+motif+for+RNA-binding+protein+HuR.">Identification of a target RNA motif for RNA-binding protein HuR.</a> Proc. Nat. Acad. Sci. 101 (9), 2987-2987 (2004).</li><li>Greenberg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+light-induced+crosslinking+of+mRNA+to+proteins.">Ultraviolet light-induced crosslinking of mRNA to proteins.</a> Nucl. Acids Res. 6 (2), 715-715 (1979).</li><li>Wagenmakers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+mRNA+to+proteins+by+irradiation+of+intact+cells+with+ultraviolet+light.">Cross-linking of mRNA to proteins by irradiation of intact cells with ultraviolet light.</a> Eur. J. Biochem. 112 (2), 323-323 (1980).</li><li>Mayrand, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+nuclear+ribonucleoprotein:+identification+of+proteins+in+contact+with+poly(A)++heterogeneous+nuclear+RNA+in+living+HeLa+cells.">Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.</a> The Journal of Cell Biology. 90 (2), 380-380 (1981).</li><li>Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+heterogeneous+nuclear+RNA-protein+complexes+in+vivo+with+monoclonal+antibodies.">Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.</a> Mol. Cell. Biol. 4 (6), 1104-11 (1984).</li><li>Adam, S. A., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+proteins+associated+with+mRNA+and+hnRNA+in+infected+HeLa+cells.">Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.</a> J. Virol. 61 (10), 3276-3276 (1987).</li><li>Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302 (5648), 1212-1212 (2003).</li><li>Licatalosi, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456 (7221), 464-464 (2008).</li><li>Yeo, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+code+for+the+FOX2+splicing+regulator+revealed+by+mapping+RNA-protein+interactions+in+stem+cells.">An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells.</a> Nat. Struct. Mol. Biol. 16 (2), 130-130 (2009).</li><li>Sanford, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+factor+SFRS1+recognizes+a+functionally+diverse+landscape+of+RNA+transcripts.">Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.</a> Genome Res. 19 (3), 381-381 (2009).</li><li>Granneman, S. <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+protein+binding+sites+on+U3+snoRNA+and+pre-rRNA+by+UV+cross-linking+and+high-throughput+analysis+of+cDNAs.">Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs.</a> Proc. Nat. Acad. Sci. , (2009).</li><li>Guil, S., Caceres, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+RNA-binding+protein+hnRNP+A1+is+required+for+processing+of+miR-18a.">The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a.</a> Nat. Struct. Mol. Biol. 14 (7), 591-591 (2007).</li><li>Chi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argonaute+HITS-CLIP+decodes+microRNA-mRNA+interaction+maps.">Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.</a> Nature. 460 (7254), 479-479 (2009).</li><li>Zisoulis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+discovery+of+endogenous+Argonaute+binding+sites+in+Caenorhabditis+elegans.">Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.</a> Nat. Struct. Mol. Biol. , (2010).</li><li>Hafner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. , (2010).</li><li>Hafner, M. <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+microRNAs+and+other+small+regulatory+RNAs+using+cDNA+library+sequencing.">Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.</a> Methods. 44 (1), 3-3 (2008).</li><li>Andrus, A., Kuimelis, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+composition+analysis+of+nucleosides+using+HPLC.">Base composition analysis of nucleosides using HPLC.</a> Current Protocols in Nucleic Acid Chemistry. Chapter 10 (Unit 10.6), (2001).</li></ol>

TT هو أحد مؤسسي والمستشار العلمي للصيدلة Alnylam ومستشار التداوي Regulus.

مخازن والكواشف نمو الخلايا المتوسطة HEK293 <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> DMEM </li><li style=";text-align:right;direction:rtl"> 10 ٪ FBS </li><li style=";text-align:right;direction:rtl"> 2 مم L - الجلوتامين </li><li style=";text-align:right;direction:rtl"> 100 U / مل البنسلين </li><li style=";text-align:right;direction:rtl"> 100 U / مل الستربتوميسين </li><li style=";text-align:right;direction:rtl"> 100 ميكروغرام / مل هيغروميسين </li><li style=";text-align:right;direction:rtl"> 15 ميكروغرام / مل blasticidin </li></ul> 4 - thiouridine حل سهم (1 م) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 260.27 ملغ 4 - thiouridine </li><li style=";text-align:right;direction:rtl"> 1 مل DMSO </li></ul> Doxycyclin الأسهم (10 ملغ / مل) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 10 ملغ doxycyclin </li><li style=";text-align:right;direction:rtl"> 1 مل DMSO </li></ul> 1X العازلة NP40 تحلل إعداد مخزون من دون العازلة 5X DTT ومثبطات الأنزيم البروتيني. إضافة DTT ومثبط البروتياز مباشرة أمام التجربة. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 HEPES ملم ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 150 ملم بوكل </li><li style=";text-align:right;direction:rtl"> 2 مم EDTA </li><li style=";text-align:right;direction:rtl"> 1 ملم ناف </li><li style=";text-align:right;direction:rtl"> 0.5 ٪ (V / V) NP40 </li><li style=";text-align:right;direction:rtl"> 0.5 مم DTT </li><li style=";text-align:right;direction:rtl"> مثبط البروتياز EDTA كاملة خالية من الكوكتيل (روش) </li></ul> سيترات ، فوسفات العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 4.7 غرام / لتر حامض الستريك </li><li style=";text-align:right;direction:rtl"> 9.2 غرام / لتر نا 2 4 هبو </li><li style=";text-align:right;direction:rtl"> الرقم الهيدروجيني 5،0 </li></ul> IP غسل العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي HEPES - كوه ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 300 ملم بوكل </li><li style=";text-align:right;direction:rtl"> 0.05 ٪ (V / V) NP40 </li><li style=";text-align:right;direction:rtl"> 0.5 مم DTT (إضافة مباشرة قبل التجربة) </li><li style=";text-align:right;direction:rtl"> مثبط البروتياز EDTA كاملة خالية من الكوكتيل (روش) (إضافة مباشرة قبل التجربة) </li></ul> عالية الملح يغسل العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي HEPES - كوه ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 500 ملم بوكل </li><li style=";text-align:right;direction:rtl"> 0.05 ٪ (V / V) NP40 </li><li style=";text-align:right;direction:rtl"> 0.5 مم DTT (إضافة مباشرة قبل التجربة) </li><li style=";text-align:right;direction:rtl"> مثبط البروتياز EDTA كاملة خالية من الكوكتيل (روش) (إضافة مباشرة قبل التجربة) </li></ul> نزع الفسفات العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.9 </li><li style=";text-align:right;direction:rtl"> 100 مم كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 10 ملي MgCl 2 </li><li style=";text-align:right;direction:rtl"> 1 ملم DTT </li></ul> الفوسفاتيز غسل العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 20 ملي EGTA </li><li style=";text-align:right;direction:rtl"> 0.5 ٪ (V / V) NP40 </li></ul> عديد النوكليوتيد كيناز (PNK) العازلة دون DTT <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 50 ملي كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 10 ملي MgCl 2 </li></ul> PNK العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 50 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 50 ملي كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 10 ملي MgCl 2 </li><li style=";text-align:right;direction:rtl"> 5 ملم DTT </li></ul> SDS PAGE العازلة تحميل <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 10 ٪ الجلسرين (V / V) </li><li style=";text-align:right;direction:rtl"> 50 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 6.8 </li><li style=";text-align:right;direction:rtl"> 2 مم EDTA </li><li style=";text-align:right;direction:rtl"> 2 ٪ SDS (ث / ت) </li><li style=";text-align:right;direction:rtl"> 100 ملي DTT </li><li style=";text-align:right;direction:rtl"> 0.1 ٪ الأزرق bromophenol </li></ul> 2X بروتيناز K العازلة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 100 ملي تريس ، حمض الهيدروكلوريك ، ودرجة الحموضة 7.5 </li><li style=";text-align:right;direction:rtl"> 150 مم كلوريد الصوديوم </li><li style=";text-align:right;direction:rtl"> 12.5 ملم EDTA </li><li style=";text-align:right;direction:rtl"> 2 ٪ (W / V) SDS </li></ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

محاضر RNA تخضع للتنظيم واسعة posttranscriptional التي توسطت فيها العديد من البروتينات الحمض النووي الريبي ملزم عبر التمثيل (الممارسات التجارية التقييدية). هنا نقدم طريقة للتعميم لتحديد بدقة وعلى نطاق واسع transcriptome - RNA المواقع ملزمة الممارسات التجارية التقييدية.

PAR كليب -- وسيلة لتحديد نطاق Transcriptome مواقع الربط من البروتينات الحمض النووي الريبي تجليد

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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