PAR-CLIP
(photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) is a
biochemical
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology an ...
method for identifying the binding sites of cellular
RNA-binding protein
RNA-binding proteins (often abbreviated as RBPs) are proteins that bind to the double or single stranded RNA in cells and participate in forming ribonucleoprotein complexes.
RBPs contain various structural motifs, such as RNA recognition motif ( ...
s (RBPs) and
microRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of
ribonucleoside A ribonucleoside is a type of nucleoside including ribose as a component.
One example of a ribonucleoside is cytidine
Cytidine (symbol C or Cyd) is a nucleoside molecule that is formed when cytosine is attached to a ribose ring (also known as a ...
analogs that are
photoreactive, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG), into nascent RNA transcripts by living cells. Irradiation of the cells by
ultraviolet
Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nanometer, nm (with a corresponding frequency around 30 Hertz, PHz) to 400 nm (750 Hertz, THz), shorter than that of visible light, but longer than ...
light of 365 nm
wavelength
In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats.
It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tro ...
induces efficient
crosslinking Cross-linking may refer to
*Cross-link
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers ca ...
of photoreactive nucleoside–
labeled cellular RNAs to interacting RBPs.
Immunoprecipitation
Immunoprecipitation (IP) 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 samp ...
of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
library and is
deep sequenced using
next-generation sequencing Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation s ...
technology.
Recently, PAR-CLIP have been applied to determine the transcriptome-wide binding sites of several known RBPs and microRNA-containing ribonucleoprotein complexes at high resolution.
Similar methods
*
CLIP-Seq High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to ...
, a similar method for identifying the binding sites of cellular RNA-binding proteins (RBPs)
or RNA modification sites
using UV light to cross-link RNA to RBPs without the incorporation of photoactivatable groups into RNA.
References
External links
starBase database decoding miRNA-mRNA, miRNA-
lncRNA
Long non-coding RNAs (long ncRNAs, lncRNA) are a type of RNA, generally defined as transcripts more than 200 nucleotides that are not translated into protein. This arbitrary limit distinguishes long ncRNAs from small non-coding RNAs, such as mic ...
, miRNA-sncRNA, miRNA-circRNA, miRNA-
pseudogene
Pseudogenes are nonfunctional segments of DNA that resemble functional genes. Most arise as superfluous copies of functional genes, either directly by DNA duplication or indirectly by Reverse transcriptase, reverse transcription of an mRNA trans ...
, protein-lncRNA, protein-ncRNA interactions and
ceRNA
Cerna may refer to:
Populated places
* Cerna, Croatia, Vukovar-Syrmia County, Croatia
* Černá (Žďár nad Sázavou District), Czech Republic
* Černá, Semily District, Czech Republic
* Cerna, Tulcea, Romania
* A village in Vaideeni Commune, V ...
networks from PAR-CLIP(
CLIP-Seq High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to ...
,
HITS-CLIP High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to ...
,
iCLIP
iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPrecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, which uses UV light to covalently bind proteins and RNA molecules to identify RN ...
) data, and '
TargetScan''
, PicTar, RNA22, miRanda and PITA microRNA target sites.
BIMSB doRiNA database a database for exploring protein-RNA, microRNA-target interactions from PAR-CLIP,
CLIP-Seq High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to ...
,
HITS-CLIP High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is a genome-wide means of mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to ...
,
iCLIP
iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPrecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, which uses UV light to covalently bind proteins and RNA molecules to identify RN ...
data, and PICTAR microRNA target site predictions.
miRTarCLIP A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing.
dCLIP dCLIP is a Perl program for discovering differential binding regions in two comparative CLIP-Seq (HITS-CLIP, PAR-CLIP or iCLIP) experiments.
PARalyzer PARalyzer is an algorithm that generates a high resolution map of interaction sites between RNA-binding proteins and their targets. The algorithm utilizes the deep sequencing reads generated from PAR-CLIP experiments.
{{DEFAULTSORT:Clip-Seq
Biochemistry detection methods
Genetics techniques
Protein methods
RNA