ICLIP

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iCLIP [1] [2] [3] (individual-nucleotide resolution crossLinking and immunoprecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, [4] which uses UV light to covalently bind proteins and RNA molecules to identify RNA binding sites of proteins. This crosslinking step has generally less background than standard RNA immunoprecipitation (RIP) protocols, because the covalent bond formed by UV light allows RNA to be fragmented, followed by stringent purification, and this also enables CLIP to identify the positions of protein-RNA interactions. [5] As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes by stringent washing during immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. The labelled protein-RNA complexes are then visualised for quality control, excised from nitrocellulose, and treated with proteinase to release the RNA, leaving only a few amino acids at the crosslink site of the RNA. [6]

The RNA is then reverse transcribed, causing most cDNAs to truncate at the crosslink site, and the key innovation and unique feature in the development of iCLIP was to enable such truncated cDNAs to be PCR amplified and sequenced using a next-generation sequencing platform. iCLIP also added a random sequence (unique molecular identifier, UMI) along with experimental barcodes to the primer used for reverse transcription, thereby barcoding unique cDNAs to minimise any errors or quantitative biases of PCR, and thus improving the quantification of binding events. Enabling amplification of truncated cDNAs led to identification of the sites of RNA-protein interactions at high resolution by analysing the starting position of truncated cDNAs, as well as their precise quantification using UMIs with software called "iCount". [1] All these innovations of iCLIP were adopted by later variants of CLIP [6] such as eCLIP [7] and irCLIP. [8] An additional approach to identify protein-RNA crosslink sites is the mutational analysis of read-through cDNAs, such as nucleotide transitions in PAR-CLIP, [9] or other types of errors that can be introduced by reverse transcriptase when it reads through the crosslink site in standard HITS-CLIP method with the Crosslink induced mutation site (CIMS) analysis. [10]

The quantitative nature of iCLIP enabled pioneering comparison across samples at the level of full RNAs, [11] or to study competitive binding of multiple RNA-binding proteins [12] or subtle changes in binding of a mutant protein at the level of binding peaks. [13] An improved variant of iCLIP (iiCLIP) was recently developed to improve the efficiency and convenience of cDNA library preparation, for example by enzymatically removing adaptor after ligation to minimise artefacts caused by adaptor carry-over, introducing the non-radioactive visualisation of the protein-RNA complex (as done originally by irCLIP [8] ), increasing efficiency of ligation, proteinase and reverse transcription reactions, and enabling bead-based purification of cDNAs. [14]

Analysis of CLIP sequencing data benefits from use of customised computational software, much of which is available as part of the Nextflow pipeline for CLIP analysis, and specialised software is available for rapid demultiplexing of complex multiplexed libraries, [15] comparative visualisation of crosslinking profiles across RNAs, [16] identification of the peaks of clustered protein-RNA crosslink sites, and identification of sequence motifs enriched around prominent crosslinks. [17] Moreover, iMaps provides a free CLIP analysis web platform and well-curated community database to facilitate studies of RNA regulatory networks across organisms, with a backend based on the Nextflow pipeline. It is applicable to the many variant protocols of CLIP (such as iCLIP, eCLIP, etc), and can be used to analyse unpublished data in a secure manner, or to obtain public CLIP data in a well-annotated format, along with various forms of quality control, visualisation and comparison. Questions on the experimental and computational challenges are collated on the Q&A CLIP Forum.

Related Research Articles

<span class="mw-page-title-main">Transcription (biology)</span> Process of copying a segment of DNA into RNA

Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins produce messenger RNA (mRNA). Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs).

<span class="mw-page-title-main">Spliceosome</span> Molecular machine that removes intron RNA from the primary transcript

A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specific proteins to form a small nuclear ribonucleoprotein complex, which in turn combines with other snRNPs to form a large ribonucleoprotein complex called a spliceosome. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material from the initial film and sends the cleaned-up version to the director for the final cut.

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 sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

<span class="mw-page-title-main">Nucleoprotein</span> Type of protein

Nucleoproteins are proteins conjugated with nucleic acids. Typical nucleoproteins include ribosomes, nucleosomes and viral nucleocapsid proteins.

RNA-binding proteins 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 (RRM), dsRNA binding domain, zinc finger and others. They are cytoplasmic and nuclear proteins. However, since most mature RNA is exported from the nucleus relatively quickly, most RBPs in the nucleus exist as complexes of protein and pre-mRNA called heterogeneous ribonucleoprotein particles (hnRNPs). RBPs have crucial roles in various cellular processes such as: cellular function, transport and localization. They especially play a major role in post-transcriptional control of RNAs, such as: splicing, polyadenylation, mRNA stabilization, mRNA localization and translation. Eukaryotic cells express diverse RBPs with unique RNA-binding activity and protein–protein interaction. According to the Eukaryotic RBP Database (EuRBPDB), there are 2961 genes encoding RBPs in humans. During evolution, the diversity of RBPs greatly increased with the increase in the number of introns. Diversity enabled eukaryotic cells to utilize RNA exons in various arrangements, giving rise to a unique RNP (ribonucleoprotein) for each RNA. Although RBPs have a crucial role in post-transcriptional regulation in gene expression, relatively few RBPs have been studied systematically.It has now become clear that RNA–RBP interactions play important roles in many biological processes among organisms.

<span class="mw-page-title-main">TATA-binding protein</span> Protein-coding gene in the species Homo sapiens

The TATA-binding protein (TBP) is a general transcription factor that binds specifically to a DNA sequence called the TATA box. This DNA sequence is found about 30 base pairs upstream of the transcription start site in some eukaryotic gene promoters.

Cross-linking and immunoprecipitation is a method used in molecular biology that combines UV crosslinking with immunoprecipitation in order to identify RNA binding sites of proteins on a transcriptome-wide scale, thereby increasing our understanding of post-transcriptional regulatory networks. CLIP can be used either with antibodies against endogenous proteins, or with common peptide tags or affinity purification, which enables the possibility of profiling model organisms or RBPs otherwise lacking suitable antibodies.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are complexes of RNA and protein present in the cell nucleus during gene transcription and subsequent post-transcriptional modification of the newly synthesized RNA (pre-mRNA). The presence of the proteins bound to a pre-mRNA molecule serves as a signal that the pre-mRNA is not yet fully processed and therefore not ready for export to the cytoplasm. Since most mature RNA is exported from the nucleus relatively quickly, most RNA-binding protein in the nucleus exist as heterogeneous ribonucleoprotein particles. After splicing has occurred, the proteins remain bound to spliced introns and target them for degradation.

<span class="mw-page-title-main">Crosslinking of DNA</span> Phenomenon in genetics

In genetics, crosslinking of DNA occurs when various exogenous or endogenous agents react with two nucleotides of DNA, forming a covalent linkage between them. This crosslink can occur within the same strand (intrastrand) or between opposite strands of double-stranded DNA (interstrand). These adducts interfere with cellular metabolism, such as DNA replication and transcription, triggering cell death. These crosslinks can, however, be repaired through excision or recombination pathways.

<span class="mw-page-title-main">FANCL</span> Protein-coding gene in the species Homo sapiens

E3 ubiquitin-protein ligase FANCL is an enzyme that in humans is encoded by the FANCL gene.

<span class="mw-page-title-main">RRBP1</span> Protein-coding gene in the species Homo sapiens

Ribosome-binding protein 1, also referred to as p180, is a protein that in humans is encoded by the RRBP1 gene.

<span class="mw-page-title-main">RBM9</span> Protein-coding gene in the species Homo sapiens

RNA binding motif protein 9 (RBM9), also known as Rbfox2, is a protein which in humans is encoded by the RBM9 gene.

<span class="mw-page-title-main">NOVA1</span> Protein-coding gene in the species Homo sapiens

RNA-binding protein Nova-1 is a protein that in humans is encoded by the NOVA1 gene.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

<span class="mw-page-title-main">RNA immunoprecipitation chip</span>

RIP-chip is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of interest in vivo. It can also be used to determine relative levels of gene expression, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins.

<span class="mw-page-title-main">Chromatin immunoprecipitation</span> Genomic technique

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly define cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

PAR-CLIP is a biochemical method for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). The method relies on the incorporation of ribonucleoside 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 light of 365 nm wavelength 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 is deep sequenced using next-generation sequencing technology.

High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a variant of CLIP for genome-wide mapping protein–RNA binding sites or RNA modification sites in vivo. HITS-CLIP was originally used to generate genome-wide protein-RNA interaction maps for the neuron-specific RNA-binding protein and splicing factor NOVA1 and NOVA2; since then a number of other splicing factor maps have been generated, including those for PTB, RbFox2, SFRS1, hnRNP C, and even N6-Methyladenosine (m6A) mRNA modifications.

Unique molecular identifiers (UMIs), or molecular barcodes (MBC) are short sequences or molecular "tags" added to DNA fragments in some next generation sequencing library preparation protocols to identify the input DNA molecule. These tags are added before PCR amplification, and can be used to reduce errors and quantitative bias introduced by the amplification.

<span class="mw-page-title-main">Ribonucleoprotein Networks Analyzed by Mutational Profiling</span> Protein-RNA binding probing method

Ribonucleoprotein Networks Analyzed by Mutational Profiling (RNP-MaP) is a strategy for probing RNA-protein networks and protein binding sites at a nucleotide resolution. Information about RNP assembly and function can facilitate a better understanding of biological mechanisms. RNP-MaP uses NHS-diazirine (SDA), a hetero-bifunctional crosslinker, to freeze RNA-bound proteins in place. Once the RNA-protein crosslinks are formed, MaP reverse transcription is then conducted to reversely transcribe the protein-bound RNAs as well as introduce mutations at the site of RNA-protein crosslinks. Sequencing results of the cDNAs reveal information about both protein-RNA interaction networks and protein binding sites.

References

  1. 1 2 König, Julian; Zarnack, Kathi; Rot, Gregor; Curk, Tomaz; Kayikci, Melis; Zupan, Blaz; Turner, Daniel J.; Luscombe, Nicholas M.; Ule, Jernej (July 2010). "iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution". Nature Structural & Molecular Biology. 17 (7): 909–915. doi:10.1038/nsmb.1838. ISSN   1545-9985. PMC   3000544 . PMID   20601959.
  2. ↑ Haberman, Nejc; Huppertz, Ina; Attig, Jan; König, Julian; Wang, Zhen; Hauer, Christian; Hentze, Matthias W.; Kulozik, Andreas E.; Le Hir, Hervé; Curk, Tomaž; Sibley, Christopher R.; Zarnack, Kathi; Ule, Jernej (2017-01-16). "Insights into the design and interpretation of iCLIP experiments". Genome Biology. 18 (1): 7. doi: 10.1186/s13059-016-1130-x . ISSN   1474-760X. PMC   5240381 . PMID   28093074.
  3. ↑ König, Julian; Zarnack, Kathi; Luscombe, Nicholas M.; Ule, Jernej (18 January 2012). "Protein–RNA interactions: new genomic technologies and perspectives". Nature Reviews Genetics. 13 (2): 77–83. doi:10.1038/nrg3141. PMC   4962561 . PMID   22251872.
  4. ↑ Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaz; Darnell, Robert B. (2003-11-14). "CLIP identifies Nova-regulated RNA networks in the brain". Science. 302 (5648): 1212–1215. Bibcode:2003Sci...302.1212U. doi:10.1126/science.1090095. ISSN   1095-9203. PMID   14615540. S2CID   23420615.
  5. ↑ Hafner, Markus; Katsantoni, Maria; Köster, Tino; Marks, James; Mukherjee, Joyita; Staiger, Dorothee; Ule, Jernej; Zavolan, Mihaela (2021-03-04). "CLIP and complementary methods". Nature Reviews Methods Primers. 1 (1): 1–23. doi: 10.1038/s43586-021-00018-1 . ISSN   2662-8449. S2CID   233834798.
  6. 1 2 Lee, Flora C. Y.; Ule, Jernej (2018-02-01). "Advances in CLIP Technologies for Studies of Protein-RNA Interactions". Molecular Cell. 69 (3): 354–369. doi: 10.1016/j.molcel.2018.01.005 . ISSN   1097-4164. PMID   29395060.
  7. ↑ Van Nostrand, Eric L.; Pratt, Gabriel A.; Shishkin, Alexander A.; Gelboin-Burkhart, Chelsea; Fang, Mark Y.; Sundararaman, Balaji; Blue, Steven M.; Nguyen, Thai B.; Surka, Christine; Elkins, Keri; Stanton, Rebecca; Rigo, Frank; Guttman, Mitchell; Yeo, Gene W. (June 2016). "Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)". Nature Methods. 13 (6): 508–514. doi:10.1038/nmeth.3810. ISSN   1548-7105. PMC   4887338 . PMID   27018577.
  8. 1 2 Zarnegar, Brian J.; Flynn, Ryan A.; Shen, Ying; Do, Brian T.; Chang, Howard Y.; Khavari, Paul A. (June 2016). "irCLIP platform for efficient characterization of protein-RNA interactions". Nature Methods. 13 (6): 489–492. doi:10.1038/nmeth.3840. ISSN   1548-7105. PMC   5477425 . PMID   27111506.
  9. ↑ Hafner, Markus; Landthaler, Markus; Burger, Lukas; Khorshid, Mohsen; Hausser, Jean; Berninger, Philipp; Rothballer, Andrea; Ascano, Manuel; Jungkamp, Anna-Carina; Munschauer, Mathias; Ulrich, Alexander; Wardle, Greg S.; Dewell, Scott; Zavolan, Mihaela; Tuschl, Thomas (2010-04-02). "Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP". Cell. 141 (1): 129–141. doi:10.1016/j.cell.2010.03.009. ISSN   1097-4172. PMC   2861495 . PMID   20371350.
  10. ↑ Zhang, Chaolin; Darnell, Robert B (1 June 2011). "Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data". Nature Biotechnology. 29 (7): 607–614. doi:10.1038/nbt.1873. PMC   3400429 . PMID   21633356.
  11. ↑ Tollervey, James R.; Curk, Tomaž; Rogelj, Boris; Briese, Michael; Cereda, Matteo; Kayikci, Melis; König, Julian; Hortobágyi, Tibor; Nishimura, Agnes L.; Zupunski, Vera; Patani, Rickie; Chandran, Siddharthan; Rot, Gregor; Zupan, Blaž; Shaw, Christopher E. (April 2011). "Characterizing the RNA targets and position-dependent splicing regulation by TDP-43". Nature Neuroscience. 14 (4): 452–458. doi:10.1038/nn.2778. ISSN   1546-1726. PMC   3108889 . PMID   21358640.
  12. ↑ Zarnack, Kathi; König, Julian; Tajnik, Mojca; Martincorena, Iñigo; Eustermann, Sebastian; Stévant, Isabelle; Reyes, Alejandro; Anders, Simon; Luscombe, Nicholas M.; Ule, Jernej (2013-01-31). "Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements". Cell. 152 (3): 453–466. doi:10.1016/j.cell.2012.12.023. ISSN   1097-4172. PMC   3629564 . PMID   23374342.
  13. ↑ Hallegger, Martina; Chakrabarti, Anob M.; Lee, Flora C. Y.; Lee, Bo Lim; Amalietti, Aram G.; Odeh, Hana M.; Copley, Katie E.; Rubien, Jack D.; Portz, Bede; Kuret, Klara; Huppertz, Ina; Rau, Frédérique; Patani, Rickie; Fawzi, Nicolas L.; Shorter, James (2021-09-02). "TDP-43 condensation properties specify its RNA-binding and regulatory repertoire". Cell. 184 (18): 4680–4696.e22. doi:10.1016/j.cell.2021.07.018. ISSN   1097-4172. PMC   8445024 . PMID   34380047.
  14. ↑ Lee, Flora C. Y.; Chakrabarti, Anob M.; Hänel, Heike; Monzón-Casanova, Elisa; Hallegger, Martina; Militti, Cristina; Capraro, Federica; Sadée, Christoph; Toolan-Kerr, Patrick; Wilkins, Oscar; Turner, Martin; König, Julian; Sibley, Christopher R.; Ule, Jernej (2021-08-27). "An improved iCLIP protocol": 2021.08.27.457890. bioRxiv   10.1101/2021.08.27.457890 . doi:10.1101/2021.08.27.457890. S2CID   237356396.{{cite journal}}: Cite journal requires |journal= (help)
  15. ↑ Wilkins, Oscar G.; Capitanchik, Charlotte; Luscombe, Nicholas M.; Ule, Jernej (2021). "Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer". Wellcome Open Research. 6: 141. doi: 10.12688/wellcomeopenres.16791.1 . ISSN   2398-502X. PMC   8287537 . PMID   34286104.
  16. ↑ Chakrabarti, Anob M.; Capitanchik, Charlotte; Ule, Jernej; Luscombe, Nicholas M. (2021-09-11). "clipplotr - a comparative visualisation and analysis tool for CLIP data": 2021.09.10.459763. doi:10.1101/2021.09.10.459763. S2CID   237496009.{{cite journal}}: Cite journal requires |journal= (help)
  17. ↑ Kuret, Klara; Amalietti, Aram Gustav; Jones, D. Marc; Capitanchik, Charlotte; Ule, Jernej (2022-09-09). "Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP". Genome Biology. 23 (1): 191. doi: 10.1186/s13059-022-02755-2 . ISSN   1474-760X. PMC   9461102 . PMID   36085079.
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