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

हम उपयोगी विचार - विमर्श के लिए Tuschl प्रयोगशाला के सदस्यों को धन्यवाद. महाराष्ट्र डेस्चर Akademischer Austauschdienst (DAAD) द्वारा समर्थित है. इस काम स्विस राष्ट्रीय फंड # MZ 3100A0 - 114,001 अनुदान द्वारा समर्थित किया गया, टीटी एक HHMI अन्वेषक है, और अपनी प्रयोगशाला में काम एनआईएच GM073047 अनुदान और MH08442 और स्टार फाउंडेशन द्वारा समर्थित किया गया था.

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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>

टीटी Alnylam फार्मास्यूटिकल्स के लिए एक cofounder और वैज्ञानिक सलाहकार और Regulus चिकित्सा के लिए एक सलाहकार है.

Buffers और अभिकर्मकों विकास मध्यम HEK293 कोशिकाओं <ul><li> DMEM </li><li> 10% FBS </li><li> 2 मिमी एल Glutamine </li><li> 100 यू / मिलीलीटर पेनिसिलिन </li><li> 100 यू / मिलीलीटर स्ट्रेप्टोमाइसिन </li><li> 100 / μg मिलीलीटर hygromycin </li><li> 15 μg / मिलीलीटर blasticidin </li></ul> 4-thiouridine शेयर समाधान (एम 1) <ul><li> 260.27 मिलीग्राम 4-thiouridine </li><li> 1 मिलीलीटर DMSO </li></ul> Doxycyclin स्टॉक (10 मिलीग्राम / एमएल) <ul><li> 10 मिलीग्राम doxycyclin </li><li> 1 मिलीलीटर DMSO </li></ul> 1x NP40 lysis बफर डीटीटी और protease inhibitors के बिना 5x बफर के एक शेयर की तैयारी. सीधे प्रयोग से पहले डीटीटी और protease अवरोध करनेवाला जोड़ें. <ul><li> 50 मिमी HEPES, 7.5 पीएच </li><li> 150 मिमी KCl </li><li> 2 मिमी EDTA </li><li> 1 मिमी NAF </li><li> 0.5% (v / v) NP40 </li><li> 0.5 मिमी डीटीटी </li><li> पूरा EDTA मुक्त protease अवरोध करनेवाला कॉकटेल (Roche) </li></ul> साइट्रेट फास्फेट - बफर <ul><li> 4.7 छ / एल साइट्रिक एसिड </li><li> 9.2 छ / एल ना 2 4 HPO </li><li> पीएच 5.0 </li></ul> आईपी ​​- धोने बफर <ul><li> 50 मिमी HEPES KOH, 7.5 पीएच </li><li> 300 मिमी KCl </li><li> 0.05% (v / v) NP40 </li><li> 0.5 मिमी डीटीटी (प्रयोग से पहले सीधे जोड़ने) </li><li> पूरा EDTA मुक्त protease अवरोध करनेवाला (Roche) कॉकटेल (प्रयोग से पहले सीधे जोड़ने) </li></ul> उच्च नमक धोने बफर <ul><li> 50 मिमी HEPES KOH, 7.5 पीएच </li><li> 500 मिमी KCl </li><li> 0.05% (v / v) NP40 </li><li> 0.5 मिमी डीटीटी (प्रयोग से पहले सीधे जोड़ने) </li><li> पूरा EDTA मुक्त protease अवरोध करनेवाला (Roche) कॉकटेल (प्रयोग से पहले सीधे जोड़ने) </li></ul> Dephosphorylation बफर <ul><li> 50 मिमी Tris - एचसीएल, 7.9 पीएच </li><li> 100 मिमी NaCl </li><li> 10 मिमी 2 MgCl </li><li> 1 मिमी डीटीटी </li></ul> फॉस्फेट धोने बफर <ul><li> 50 मिमी Tris - एचसीएल, 7.5 पीएच </li><li> 20 मिमी EGTA </li><li> 0.5% (v / v) NP40 </li></ul> डीटीटी बिना polynucleotide kinase बफर (PNK) <ul><li> 50 मिमी Tris - एचसीएल, 7.5 पीएच </li><li> 50 मिमी NaCl </li><li> 10 मिमी 2 MgCl </li></ul> PNK बफर <ul><li> 50 मिमी Tris - एचसीएल, 7.5 पीएच </li><li> 50 मिमी NaCl </li><li> 10 मिमी 2 MgCl </li><li> 5 मिमी डीटीटी </li></ul> एसडीएस PAGE लोड हो रहा है बफर <ul><li> 10% ग्लिसरॉल (v / v) </li><li> 50 मिमी Tris - एचसीएल, 6.8 पीएच </li><li> 2 मिमी EDTA </li><li> 2% एसडीएस (w / v) </li><li> 100 मिमी डीटीटी </li><li> 0.1% bromophenol नीले </li></ul> 2x proteinase कश्मीर बफर <ul><li> 100 मिमी Tris - एचसीएल, 7.5 पीएच </li><li> 150 मिमी NaCl </li><li> 12.5 मिमी EDTA </li><li> 2% (w / v) एसडीएस </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

शाही सेना टेप है कि पार अभिनय शाही सेना बाध्यकारी प्रोटीन (RBPs) की एक भीड़ द्वारा मध्यस्थता है व्यापक posttranscriptional विनियमन के अधीन हैं. यहाँ हम एक generalizable विधि और ठीक transcriptome की व्यापक पैमाने पर RBPs की शाही सेना बाध्यकारी साइटों की पहचान उपस्थित थे.

बराबर क्लिप - एक विधि transcriptome चौड़ा शाही सेना बंधनकारी प्रोटीन की बाइंडिंग साइटें पहचानें

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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