Table 1.
Technologies for studying post-transcriptional control of the cancer genome
| Technology | Variations | Description |
|---|---|---|
| Translational profiling | Polysome microarray | Translational profiling that uses classic microarray technology to analyse mRNAs associated with actively translating ribosomes isolated by sucrose gradient fraction234 |
| Ribosome profiling | Deep sequencing of ribosome-protected mRNA fragments as a genome-wide measure of translation with subcodon resolution235 | |
| Proximity-specific ribosome profiling | Ribosome profiling using a spatially restricted biotin ligase and biotin-acceptor-tagged ribosomes as a strategy to look at proximity-specific translation236 | |
| RNA–protein interaction network analysis | Crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-Seq) | Also known as high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), a method that uses high-throughput sequencing of RNAs isolated by UV crosslinking and immunoprecipitation to map genome-wide binding sites of a given protein within a 30-nucleotide region of RNA237 |
| Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) | An alternative to CLIP-Seq that uses PAR analogues, such as 4-thiouridine, added to cell culture to enable enhanced crosslinking and precise mapping of RBP-binding sites by scoring for crosslinking-induced thymidine to cytidine transitions238 | |
| Individual-nucleotide-resolution UV crosslinking and immunoprecipitation (iCLIP) | An adaptation of CLIP-Seq that enables improved individual-nucleotide resolution by using an adaptor that exploits the tendency of cDNAs to prematurely truncate before the crosslinked nucleotide239 | |
| RBP interactome capture | A technique using either UV or PAR crosslinking of RNA–protein interactions followed by oligo(dT) purification of mRNAs and mass spectrometry (MS) to identify the full repertoire of RBPs within a cell113 | |
| Comprehensive identification of RBPs by MS (ChIRP-MS) | A method for identifying the specific subset of proteins associated with a particular RNA using formaldehyde crosslinking of protein–RNA interactions, followed by RNA purification using tiled biotinylated probes specific for the RNA of interest, and MS to identify bound proteins240 | |
| RNA antisense purification with MS (RAP-MS) | An approach similar to ChIRP-MS that uses UV crosslinking and stable isotope labelling by amino acids in culture (SILAC) to perform quantitative MS of RNA–protein interactions241 | |
| RNA–RNA interaction network analysis | Crosslinking, ligation and sequencing of hybrids (CLASH) | A high-throughput method to identify RNA–RNA interactions associated with mRNPs based on purification of UV-crosslinked complexes, intra- and intermolecular ligation of RNA–RNA interactions, and sequencing of hybrid RNAs242 |
| RNA structure analysis | Parallel analysis of RNA structure (PARS) | A method for analysing RNA structure genome-wide at single-nucleotide resolution by parallel deep sequencing of RNA fragments generated by treatment with various structure-specific endonucleases243 |
| Fragmentation sequencing (Frag-Seq) | Genome-wide analysis to identify RNA structure based on high-throughput sequencing of RNA fragments produced by the cleavage of single-stranded nucleic acids by nuclease P1 (REF. 244) | |
| Selective 2-hydroxyl acylation analysed by primer extension (SHAPE) | This standard analysis of RNA secondary structure, which uses primer extension to measure 2′-hydroxyl group reactivity with chemical probes, has recently been improved by the development of new probes enabling its application in living cells245 | |
| SHAPE-Seq | A high-throughput version of SHAPE that enables genome-wide analysis of RNA structure by using multiplexed paired-end deep sequencing of primer extension products246 | |
| In vivo click-SHAPE (icSHAPE) | An adaptation of SHAPE that combines genome-wide profiling with in vivo click selective 2′-hydroxyl acylation to overcome previous limitations in chemical probes that precluded analysis of all four nucleotides247 | |
| RNA hybrid and individual-nucleotide-resolution UV CLIP (hiCLIP) | A modified iCLIP procedure that is similar to CLASH but which uses an adaptor in the ligation step enabling it to better resolve RNA–RNA interactions and predict secondary structure248 | |
| RNA modification profiling | m6A profiling | Techniques such as Me-RIPseq134 and m6A-Seq136 measure N6-methyladenosine modifications on RNA by high-throughput sequencing of transcripts captured using an antibody specific for the modification |
| m5C profiling | The recent development of affinity purification-based high-throughput sequencing methods for profiling RNA 5-methylcytosine modifications — such as 5-azacytidine RNA immunoprecipitation (Aza-IP)249 and methylation individual-nucleotide-resolution CLIP (miCLIP)250 — provide some advantages over bisulfite sequencing approaches251, such as the enhanced ability to detect modification on less abundant RNAs | |
| Pseudo-Seq, PSI-Seq and Ψ-Seq | High-throughput sequencing methods for genome-wide identification of RNA pseudouridylation using CMC to modify pseudouridines and generate a block in reverse transcription137,139,140 | |
| N3-CMC-enriched pseudouridine sequencing (Ceu-Seq) | An approach similar to Pseudo-Seq that uses a CMC derivative modified by click chemistry to add biotin, enabling the enrichment of pseudouridine-containing RNAs by biotin pulldown before sequencing138 | |
| Quantitative MS | Optimized MS approaches have been developed for measuring pseudouridine levels at select rRNA sites226, for determining global changes in the full range of tRNA modifications252 and for evaluating global changes in nucleoside modifications across all species of RNA253 | |
| tRNA profiling | tRNA microarray | Chemical ligation-based microarrays have been developed for profiling tRNA isoacceptors at single-nucleotide resolution141, improving on previous hybridization-based microarrays that can only distinguish tRNAs differing by at least eight nucleotides150 |
| tRNA high-throughput sequencing | Recent adaptations of RNA-Seq such as: DM-tRNA-seq254 and ARM-seq162 have been developed to overcome limits in sequencing caused by RNA modifications and structure; these techniques not only enable quantitative analysis of tRNA expression but can also provide dynamic measurements of specific tRNA methylations |
ARM-seq, AlkB-facilitated RNA methylation sequencing; CMC, N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate; DM-tRNA-seq, demethylase-thermostable group II intron RT tRNA sequencing; mRNP, messenger ribonucleoprotein; RBP, RNA-binding protein.