To understand how RNPs regulate the life cycle of mRNAs in neurons, and how this can go wrong in diseases, we need to be able to monitor RNP assembly and dynamics.
For this purpose, we have developed methods to obtain detailed maps of protein-RNA and RNA-RNA interactions in cells and tissues.
We developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to quantify protein-RNA interactions in the whole transcriptome. We collaborate with Nick Luscombe to develop computational methods for analysis and quality control of iCLIP data. We showed that most cDNAs in iCLIP truncate at crosslink sites, and in collaboration with Tomaž Curk, we developed iCount, a Python code and associated command-line interface (CLI), which are available from GitHub. iCount maps the iCLIP data, defines the peaks of high-occupancy RNA binding sites, annotate the data, identify enriched sequence motifs, and analyse the position-dependent binding patterns, or RNA maps. This code is the basis for the web server iMaps, which enables a streamlined analysis of data produced by iCLIP and its variant methods, and is available for general use.
To understand the assembly of RNPs, it is also crucial to identify the RNA-RNA contacts that form between and within RNAs, because RNA structure has an important contribution to the formation of RNP. For this purpose, developed a technique called hiCLIP (or hybrid iCLIP), which identifies the connections that hook sections of RNAs together, referred to as RNA duplexes. This identified RNA duplexes between different regions of the same RNA, as well as interactions between different RNAs, such as long-noncoding RNAs and mRNAs. We were amazed to find that these duplexes often hook together very distant parts of mRNA molecules, and these duplexes interact with the double-stranded RNA binding protein Staufen 1. In collaboration with the Luscombe group, we also showed that these RNA duplexes have less genetic variation in humans than surrounding areas of the mRNA, indicating that mutations could cause disease by disrupting the structure of mRNAs.