TABLE 2.
Large-Scale Methods for the Discovery of RBP/RNP RNA Targets
| Method | Goal | Description | Selection of RBPs Analyzed |
|---|---|---|---|
| Non-crosslinking methods | |||
| RIP-Chip | Target gene identification | IP of epitope tagged or endogenous RBP/RNP to isolate-associated RNAs. Isolated RNAs are analyzed by microarray. RBP/RNP targets are defined by calculating and scoring according to enrichment of IP′d transcripts over control expression values. | For comprehensive list, see Table 1 in Ref 33. |
| RNA IP and high throughput sequencing (RIP-Seq) | Target gene identification | Same procedure as RNA IP followed by microarray analysis (RIP-Chip); IP′d RNA is quantified by RNAseq. RBP/RNP targets are defined, similar to RIP-Chip, by calculating enrichment scores over control. | TDP-43,34 LIN-28,35 Polycomb proteins32 |
| Photocrosslinking methods | |||
| CLIP | Target site identification; definition of RRE | Ultraviolet (UV) 254 photocrosslinking of RNA to RBPs in live cells or tissues prior to lysis. Crosslinked RNA is trimmed by RNases and RNA– protein complexes are fractionated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Crosslinked RNAs are isolated, 3′ and 5′ adapters for RT and polymerized chain reaction ligated. The cDNA library is inserted into plasmid for bacterial transformation, cloning, and sequencing. | Nova,36 SF2,37 CUGBP1,38 Rsr,39 MSY2,40 SAM68,41 hnRNP A142 |
| HiTS-CLIP | Target site identification; definition of RRE | Similar to standard CLIP, however, cDNAs are deep sequenced (454 or Solexa). Overlapping sequence reads are clustered and RBP/RNP targets are defined based on enrichment over negative controls. | TDP-43,43,44 Nova,45 AGO2,46,47 Alg-1,48 SFRS1,49 FOX2,50 PTB,51 Khd152 |
| iCLIP | Target site identification; definition of RRE | Crosslinking procedures similar to CLIP. Partial cDNAs, generated from stalled RT of crosslinked RNAs, are circularized and ultimately deep sequenced. RBP/RNP targets are similarly defined as in HiTS-CLIP. Sites of crosslinking are interpreted as the (−1) position a sequence read maps to (indicating the putative RT stall sites). | hnRNP C,53 TIA1, TIAL154 |
| iCLAP | Target site identification; definition of RRE | Similar to iCLIP except that the RBP is Strep- and polyhistidine epitope tagged. Streptavidin beads are used in the first purification step. Immobilized metal-ion affinity chromatography (IMAC) under denaturing conditions is performed as a secondary purification. Isolated RNAs are converted into cDNA and sequenced similar to iCLIP. | TIA1, TIAL154 |
| CRAC | Target site identification | Similar in concept to CLAP, where a tandem affinity purification protocol is used. RBPs are engineered to contain C-terminal 6X-histidine, tobacco etch virus (TEV) protease site, and Protein A tags. Immunoglobulin G beads are used as a first purification step, followed by TEV protease treatment to elute crosslinked RNA–protein complexes. IMAC is performed under denaturing conditions as a secondary purification. Individual RNAs were analyzed by northern blot or sequenced after amplification with gene-specific primers. | Prp43,55 U3 snoRNA-binding sites of Nop1, Nop56, Nop58, And Rrp956 |
| Photoactivatable ribonucleoside- enhanced CLIP | Target site identification; definition of RRE | Incubation of live cells with photoactivatable ribonucleosides (4-thiouridine and 6-thioguanosine) that are incorporated into nascent transcripts. UV- 365 photocrosslinking of RNA to proteins prior to lysis. IP of epitope tagged or endogenous RBP/RNP to isolate-associated RNAs. cDNA library preparation of isolated RNAs. Putative RBP/RNP target sites are scored by frequency of crosslinking evidence, seen as a characteristic T2C (or G2A) nucleotide mutation. | ELAVL1,57,58 QKI,59 IGF2BP1–3,59AGO1–4,59 PUM2,59 TNRC6A–C,59 FUS,60 EWSR1,60 TAF1560 In analysis: BICC1, CUGBP1, DHX9, DICER, DND1, EXPORTIN5, FMR1, FXR1, FXR2, ELAVL2–4, LIN28B, MBNL1, MOV10, NCL, P54, RBM20, RBPMS, SND1, TARBP2, TDP-43, TIA1,TSN, TSNAX, TTP, WT1 |
CLIP, crosslinking and immunoprecipitation; HiTS-CLIP, High-throughput sequencing CLIP; iCLAP, individual nucleotide resolution crosslinking affinity purification; iCLIP, individual nucleotide resolution CLIP; IP, immunoprecipitation; RBP, RNA-binding proteins; RNPs, ribonucleoprotein complexes; RRE, RNA recognition element; RT, reverse transcription.