ClipSeq /CRAC

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It is a crosslinking method that uses UV light to cross-link RNA to Proteins without the incorporation of photoactivatable groups into RNA, followed by sequencing of the linked RNA. References Sander Granneman, Grzegorz Kudla, Elisabeth Petfalski, and David Tollervey1 Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9613-8 PMID: 19482942

Bahrami-Samani E1, Penalva LO2, Smith AD1, Uren PJ3. Leveraging cross-link modification events in CLIP-seq for motif discovery. Nucleic Acids Res. 2015 Jan;43(1):95-103. PMID: 25505146

Reported by: simonapanni

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Hi Simona,

I was having a look at your method requests and I have a question. Concerning CLIP-Seq/CRAC, it seems that hte main difference of this method from other forms of CLIP is that the proteins were pulled out via a tag that they bear, instead of being co-immunoprecipitated. The cross linking event takes place as you describe, without using photoactivable groups, etc... The RNA can be detected with northern blot afterwards, but it seems it has been applied using sequencing as well and it is still called CRAC is those instances (emphasizing the affinity purification methods over co-ip).

Now the CLIP-Seq term, also named HIST-CLIP, seems to be used more in cases where the proteins were co-ip'ed and the RNA/cDNA read via HT sequencing. It is used in high-throughput setups, allowing the identification of multiple binding sites. There is yet another method named iCLIP in which individual binding sites are detected in high-resolution, but I do not know if that's something you need.

Do you need the creation of both the CRAC andthe CLIP-Seq/HIST-CLIP methods? More important, did I get everything right? You are more familiar with the techniques, so please correct me if I am wrong.

Cheers,

Pablo.

Original comment by: pporras

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Posted on behalf of Simona Panni:

In general I was trying to reduce the number of different methods when differences are little, but, on the contrary, we can decide to be more rigorous and strictly follow the authors' definitions. This is probably better, and in this case I need all the methods you have mentioned( and I will list them again below) and it is better to divide CRAC from Clipseq even if definition are similar.

To answer to your question, if we divide clip-seq from crac it is true that the proteins used in crac are tagged because the group of Tollervey who has proposed the protocol always uses tagged proteins. Instead I wouldn' t say that CLIP-seq uses tagged proteins, but (as you said) that Clip-seq was developed few years later than Clip, and make use of new sequencing techniques. I attach a review that clearly describe the CLIP methods, I have compared it with other papers and I thinks their definitions are fitting and we could use them.

So the whole list of this kind of methods is:

CLIP (UV cross linking, Immunoprecipitation, proteinase K, RT-PCR, cloned and identified by sequence or other methods ) One of the original reference PMID: 14615540

CLIP-SEQ/Hits CLIP (UV crosslinking Immunoprecipitation, HT seq) The original ref is PMID:18978773

PAR-CLIP crosslinks RNA that has incorporated 4-thiouridine (4-SU) to proteins in vivo. The protein is immunoprecipitated and the bound RNAs are revealed by sequencing (DONE) Ref: PMID: 20371350

iCLIP Similar to Clip-Seq but the single binding nucleotide is identify by using two inversely oriented cleavable 3' adapter regions linked to a barcode which serves to identify the binding site. Ref PMID: 20601959

CRAC (UV cross linking of tagged proteins (?), Immunoprecipitation, proteinase K, RT-PCR, cloned and identified by sequence or other methods ) Ref PMID: 19482942

CLASH (RNA-RNA interactions that occur in the proximity of an RNA binding protein of interest. The RNA is crosslinked to the protein, that is immunoprecipitated. After RNAsi treatment the RNA hybrids protected from degradation by the protein are ligated and chimeras are sequenced) ref PMID 21610164 Thanks for your help and please let me know what you decide

Simona

Original comment by: pporras

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The following terms have been created:

CLIP, MI:2191 Combination of cross-linking and co-immunoprecipitation aimed to find protein-RNA interactions. The canonical method uses first a cross-linking procedure over a tissue sample or lysate, and the immunoprecipitated with specific antibodies for the protein of interest. Unspecific proteins are digested via proteinase K treatment. RNA can be then identified via Northern blotting or using RT-PCR and then sequencing of the generated cDNA. https://en.wikipedia.org/wiki/CLIP

Child of CLIP: CLIP-Seq, MI:2192 This method combines UV cross-linking and immunoprecipitation with high-throughput sequencing to identify binding sites of RNA-binding proteins. https://en.wikipedia.org/wiki/HITS-CLIP

Child of CLIP: iCLIP, MI:2193 iCLIP allows for the stringent purification of UV cross-linked protein-RNA complexes, using immunoprecipitation followed by SDS-PAGE and membrane transfer. The radiolabelled protein-RNA complexes are then excised from the membrane, and treated with proteinase to release the RNA. This leaves one or two amino acids at the RNA cross-link site. The RNA is then reverse transcribed using barcoded primers. Because reverse transcription stops prematurely at the cross-link site, iCLIP allows RNA-protein interaction sites to be identified at high resolution. https://en.wikipedia.org/wiki/ICLIP

Child of CLIP: PAR-CLIP, MI:2188 Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) is a biochemical method used for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). 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 next-generation sequencing technology. http://en.wikipedia.org/wiki/PAR-CLIP

CRAC, MI:2194 Combination of UV cross-linking and affinity purification where the protein of interest bears a tag used for pull-down or immunoprecipitation. As in CLIP, unspecific proteins are digested via proteinase K treatment and RNAs are tagged with oligonucleotide linkers. RNA can be then identified via Northern blotting or using RT-PCR and then sequencing of the generated cDNA.

Child of CRAC: CLASH, MI:2195 This method is a variation of CRAC where after combined cross-linking and affinity purification of protein-RNA complexes, RNA-RNA interactions are specifically detected. Base-paired RNA molecules can be linked together during while tagging RNA with oligonucleotides, generating chimeric RNAs that allow identification of RNA-RNA pairs.

The terms have been uploaded to the latest version of the CV and are ready for use.

Pablo.

Original comment by: pporras

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Original comment by: pporras