Huppertz, I;
Haberman, N;
Ule, J;
(2018)
‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP.
Wellcome Open Research
, 3
, Article 77. 10.12688/wellcomeopenres.14663.1.
Preview |
Text
940ce845-f97f-4dc8-a8fe-45b5397fdee3_14663_-_jernej_ule.pdf - Published Version Download (1MB) | Preview |
Abstract
We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5’-end of RNA fragments—a 5’-marker—to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins’ RNA-binding sites. Thus, cDNA start sites confidently identify a protein’s RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5’-marker.
| Type: | Article |
|---|---|
| Title: | ‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.12688/wellcomeopenres.14663.1 |
| Publisher version: | http://doi.org/10.12688/wellcomeopenres.14663.1 |
| Language: | English |
| Additional information: | Copyright: © 2018 Huppertz I et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited |
| Keywords: | Protein–RNA interactions, iCLIP, High-throughput sequencing, Polypyrimidine tract binding protein 1 (PTBP1), Eukaryotic initiation factor 4A-III (eIF4A3) |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10053013 |
Archive Staff Only
 |
View Item |
