Comparative analysis of LIN28-RNA binding sites identified at single nucleotide resolution

Abstract

ABSTRACT It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes. Interestingly, analyses by the two methods diverge in their identification of crosslinked nucleotide identity – whereas bioinformatics and sequencing analyses suggest guanine in mammalian cells, MS/MS identifies uridine. This work suggests the need for comprehensive analysis and validation of crosslinking methodologies.