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

Мы благодарим членов Tuschl лаборатории за полезные обсуждения. MH поддерживается Deutscher Akademischer Austauschdienst (DAAD). Работа выполнена при поддержке Швейцарского национального фонда, грант № 3100A0-114001 для MZ; ТТ следователь HHMI, и работать в его лаборатории была поддержана грантами NIH GM073047 и MH08442 и Старр Foundation.

<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 является соучредителем и научный советник Alnylam Фармацевтические препараты и советник Регул Therapeutics.

Буферы и реагенты Рост средний НЕК293 <ul><li> DMEM </li><li> 10% FBS </li><li> 2 мМ L-глутамина </li><li> 100 ЕД / мл пенициллина </li><li> 100 ЕД / мл стрептомицина </li><li> 100 мкг / мл гигромицину </li><li> 15 мкг / мл blasticidin </li></ul> 4-тиоуридина маточного раствора (1 М) <ul><li> 260,27 мг 4-тиоуридина </li><li> 1 мл ДМСО </li></ul> Doxycyclin акции (10 мг / мл) <ul><li> 10 мг doxycyclin </li><li> 1 мл ДМСО </li></ul> 1x NP40 лизирующего буфера Подготовить запас 5x буфера без DVB-T и ингибиторы протеазы. Добавить DTT и ингибитор протеазы непосредственно перед экспериментом. <ul><li> 50 мМ HEPES, рН 7,5 </li><li> 150 мМ KCl </li><li> 2 мМ ЭДТА </li><li> 1 мМ NaF </li><li> 0,5% (объем / объем) NP40 </li><li> 0,5 мМ DTT </li><li> полный ЭДТА без ингибитора протеазы коктейль (Roche) </li></ul> Цитрат-фосфатного буфера <ul><li> 4,7 г / л лимонной кислоты </li><li> 9,2 г / л Na 2 HPO 4 </li><li> рН 5,0 </li></ul> IP-промывочный буфер <ul><li> 50 мМ HEPES-KOH, рН 7,5 </li><li> 300 мМ KCl </li><li> 0,05% (объем / объем) NP40 </li><li> 0,5 мМ DTT (добавить непосредственно перед экспериментом) </li><li> полный ЭДТА без ингибитора протеазы коктейль (Roche) (добавить непосредственно перед экспериментом) </li></ul> Высокая-солевого буфера для промывки <ul><li> 50 мМ HEPES-KOH, рН 7,5 </li><li> 500 мМ KCl </li><li> 0,05% (объем / объем) NP40 </li><li> 0,5 мМ DTT (добавить непосредственно перед экспериментом) </li><li> полный ЭДТА без ингибитора протеазы коктейль (Roche) (добавить непосредственно перед экспериментом) </li></ul> Дефосфорилирования буфер <ul><li> 50 мМ Трис-HCl, рН 7,9 </li><li> 100 мМ NaCl </li><li> 10 мМ MgCl 2 </li><li> 1 мМ DTT </li></ul> Фосфатазы промывочный буфер <ul><li> 50 мМ Трис-HCl, рН 7,5 </li><li> 20 мМ EGTA </li><li> 0,5% (объем / объем) NP40 </li></ul> Полинуклеотидкиназы (ПНК) буфера без DTT <ul><li> 50 мМ Трис-HCl, рН 7,5 </li><li> 50 мМ NaCl </li><li> 10 мМ MgCl 2 </li></ul> PNK буфер <ul><li> 50 мМ Трис-HCl, рН 7,5 </li><li> 50 мМ NaCl </li><li> 10 мМ MgCl 2 </li><li> 5 мМ DTT </li></ul> SDS загрузки страниц буфера <ul><li> 10% глицерина (об / об) </li><li> 50 мМ Трис-HCl, рН 6,8 </li><li> 2 мМ ЭДТА </li><li> 2% SDS (м / о) </li><li> 100 мМ DTT </li><li> 0,1% бромфенола синий </li></ul> 2x протеиназы K буфера <ul><li> 100 мМ Трис-HCl, рН 7,5 </li><li> 150 мМ NaCl </li><li> 12,5 мМ ЭДТА </li><li> 2% (м / о) 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

РНК-транскрипты подвергаются обширные посттранскрипционные регулирование, которое опосредуется множеством транс действия РНК-связывающими белками (ОДП). Здесь мы представляем обобщению метода точно определить и на транскриптома масштабе РНК места связывания ОДП.

PAR-Clip - метод идентификации транскриптома всей места связывания РНК-связывающие белки

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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