'Read-through marking' reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP.

Ina Huppertz,Jernej Ule,Nejc Haberman

doi:10.12688/wellcomeopenres.14663.1

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.

Highlights

As part of the individual-nucleotide resolution cross-linking and immunoprecipitation protocol, crosslinked protein– RNA complexes are immunopurified and the RNA fragments are released by protein digestion, resulting in RNAs with a covalently bound peptide at the crosslink site (König et al, 2010; Lee & Ule, 2018)
This applies to related techniques that rely on analysis of truncated cDNAs, including among others the FAST-individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), CITS-CLIP, BrdU-CLIP, irCLIP, eCLIP and miCLIP, as reviewed recently (Lee & Ule, 2018)
To examine the characteristics of read-through cDNAs that are present in iCLIP cDNA libraries, we introduced an additional RNA ligation reaction that adds an oligonucleotide to the 5’-end of the RNA fragments (5’-marker; Figure 1a, step 3b)

Summary

Introduction

As part of the individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) protocol, crosslinked protein– RNA complexes are immunopurified and the RNA fragments are released by protein digestion, resulting in RNAs with a covalently bound peptide at the crosslink site (König et al, 2010; Lee & Ule, 2018) This is followed by reverse transcription, where in theory the RNA–peptide complex leads to the premature termination of cDNA synthesis and is indicative of a protein’s binding site. In iCLIP, the start of the truncated cDNAs should be equivalent to the position of the crosslink sites This applies to related techniques that rely on analysis of truncated cDNAs, including among others the FAST-iCLIP, CITS-CLIP, BrdU-CLIP, irCLIP, eCLIP and miCLIP, as reviewed recently (Lee & Ule, 2018). Understanding the characteristics of read-through cDNAs in iCLIP is essential, since their presence could erroneously shift the boundaries of predicted RNAbinding sites to positions upstream of their true binding sites

Methods

Results

Conclusion