UC San Diego

At any time hundreds of thousands of macromolecular interactions occur in a cell, mediating functions that maintain normal cellular activities. A fundamental problem in molecular biology is to catalog these interactions and to decipher their functional consequences. High throughput sequencing has made it possible to characterize some of these interactions rapidly, at high-resolution, and in vivo (e.g., protein-DNA binding via ChIP-Seq and protein-RNA binding via CLIP-Seq). But many interactions are not susceptible to these methods (e.g., RNA- RNA complexes, ncRNA-DNA binding, protein-protein interactions).

This thesis aims to address this gap by coupling high-throughput sequencing with proximity-ligation-based methods. In proximity ligation, spatially proximate nucleic acids ligate to one another, forming a chimeric oligonucleotide. Observation of a chimera composed of X and Y suggests that X and Y must have been near one another in the original sample. As a result, questions about spatial arrangement become questions about sequence composition, making it possible to take advantage of high-throughput sequencing. Using this general approach, we developed high-throughput technologies to study RNA interactions with different types of molecular partners: RNA, chromatin, and lipid.

In Chapter 1, I review and discuss many different high-throughput techniques to map RNA structure and RNA-RNA as well as RNA-chromatin interactions. I also provide the biological insight that can be gained from the type of data generated by the new technologies.

In Chapter 2, I describe the development of MARIO, a technology to map RNA-RNA interactions. This method produces a global map of RNA-RNA interactome and RNA structures in vivo. The information will provide roadmaps to systems level understanding of cellular regulations through RNA-RNA interactions.

In Chapter 3, I describe the development of MARGI, a technology to map RNA-chromatin interactions. Mapping RNA-chromatin interactions identify RNAs that bind to the chromatin and shed light on the functional roles of chromatin associated RNAs in gene regulations.

In Chapter 4, I describe the discovery of a new class of RNA, named cell surface RNA. These are RNAs that are expressed outside of the cell membrane. I also develop methods to isolate and identify these surface RNAs and protein binding partners of the RNAs