CLIP-Seq
Encyclopedia
CLIP-Seq, also called HITS-CLIP
, is a method in molecular biology
, used for finding which RNA
species interact with a particular RNA-binding protein
(or an RNA). It employs UV-crosslinking between RNA and the protein, followed by immunoprecipitation
with antibodies for the protein, fragmentation, high-throughput sequencing and bioinformatics
.
Recently, HITS-CLIP (CLIP-Seq) of Argonaute
has been applied to decode microRNA-target interaction maps. The application of CLIP-Seq/HITS-CLIP methods has significantly reduced the rate of false positive predictions of miRNA binding sites and has also reduced the size of the search space for miRNA target sites. Improved analysis of Argonaute HITS-CLIP now enables identification of binding sites with single nucleotide resolution.
) are:
HITS-CLIP
High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation is a genome-wide means of mapping protein–RNA binding sites in vivo...
, is a method in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, used for finding which RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
species interact with a particular RNA-binding protein
RNA-binding protein
RNA-binding proteins are proteins that bind to RNA. They bind to either double-strand or single-strand RNAs through RNA recognition motif . RNA-binding proteins may regulate the translation of RNA, and post-transcriptional events, such as RNA splicing, editing.They are cytoplasmic and nuclear...
(or an RNA). It employs UV-crosslinking between RNA and the protein, followed by immunoprecipitation
Immunoprecipitation
Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins...
with antibodies for the protein, fragmentation, high-throughput sequencing and bioinformatics
Bioinformatics
Bioinformatics is the application of computer science and information technology to the field of biology and medicine. Bioinformatics deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, software...
.
Recently, HITS-CLIP (CLIP-Seq) of Argonaute
Argonaute
Argonaute proteins are the catalytic components of the RNA-induced silencing complex , the protein complex responsible for the gene silencing phenomenon known as RNA interference . Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs , small interfering RNAs and...
has been applied to decode microRNA-target interaction maps. The application of CLIP-Seq/HITS-CLIP methods has significantly reduced the rate of false positive predictions of miRNA binding sites and has also reduced the size of the search space for miRNA target sites. Improved analysis of Argonaute HITS-CLIP now enables identification of binding sites with single nucleotide resolution.
Computational analysis
Currently there is no a standard pipeline to analyze this kind of data. All the published data have been analysed with home-made scripts.miRNA targets detection
The main steps (using at the same time degradome data Degradome sequencingDegradome sequencing
Degradome sequencing , also referred to as parallel analysis of RNA ends , is a modified 5'-rapid amplification of cDNA ends with high-throughput deep sequencing method...
) are:
- mapping CLIP-seq reads
- mapping Degradome-Seq reads
- the overlapping reads were grouped into clusters
- miRNA targets from different public databases
- miRNA–target interactions with an alignment score from CleaveLand not exceeding the cutoff threshold of 7.0
- the ClipSearch program was developed to search for 6–8-mers (8-mer, 7-mer-m8 and 7-mer-A1) (2,5) in CLIP-Seq data
- The DegradomeSearch program was developed to search Degradome-Seq clusters for nearly perfect complements of miRNA sequences
Similar methods
- PAR-CLIPPAR-CLIPPAR-CLIP is a novel method, used for identifying the binding sites of cellular RNA-binding proteins and microRNA-containing ribonucleoprotein complexes...
, for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) in tissue culture cells. - RIP-ChipRIP-ChipRIP-Chip is immunoprecipitation of an RNA-binding protein coupled to reverse transcription and a microarray. It has been used to find interactions between RNA and protein ....
, same goal and first steps, but doesn't use crosslinking and uses microarray instead of sequencing - ChIP-Seq, for finding interactions with DNA rather than RNA
- SELEXSystematic Evolution of Ligands by Exponential EnrichmentSELEX , also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands....
, a method for finding a consensus binding sequence
External links
- starBase database: a database for exploring protein-RNA and miRNA-target interactions from CLIP-Seq (HITS-CLIPHITS-CLIPHigh-throughput sequencing of RNA isolated by crosslinking immunoprecipitation is a genome-wide means of mapping protein–RNA binding sites in vivo...
, PAR-CLIPPAR-CLIPPAR-CLIP is a novel method, used for identifying the binding sites of cellular RNA-binding proteins and microRNA-containing ribonucleoprotein complexes...
) data.