UC San Diego

RNA and RNA-binding-proteins (RBPs) play essential roles in diverse cellular functions and disease-relevant pathways. Currently, the sequence-specificity of RNA-RBP interactions and the RNA binding of disease-related RBPs are under studied. Investigating RNA-RBP interaction mechanisms, both at a molecular level and the whole transcriptomic level, is thus crucial to the understanding of fundamental RNA biology and the development of potential RNA therapeutics. The primary focus of this dissertation is on RNA-RBP interaction mechanisms and novel tool development for sequence-specific RNA-targeting and endogenous RNA-RBP interaction profiling. In Chapter 1, to understand the RNA-recognition rules and to develop a designer protein toolbox for RNA targeting, mutations of an RNA-binding zinc finger (ZnF) in human ZRANB2 was systematically introduced at the key RNA-recognizing residues. RNA-bind-n-seq (RBNS) assay identified mutant ZnFs with an altered sequence specificity, preferring to bind a GGG motif instead of the GGU preferred by wild-type ZRANB2 ZnF. Further, through all-atom molecular dynamics (MD) simulations, we characterized changes in the hydrogen-bond network between the protein and RNA that underly the observed sequence specificity changes. In Chapter 2, we developed INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the current challenges in probing RBP-RNA interactions within endogenous biological contexts. Harnessing the in situ RNA labeling by the APOBEC1 enzyme directed by the nanobody-antibody recognition, this method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. Chapter 1 in this dissertation greatly deepens the understanding of RNA-ZnF molecular recognition, which is an important foundation for engineering RNA-binding ZnFs for programmable RNA targeting. The technology presented in Chapter 2, INSCRIBE, can be broadly applied in RBP-RNA interaction profiling in a variety of biological samples, bridging the critical gap in unveiling RBP roles in RNA-related diseases within endogenous biological contexts such as developmental studies, animal disease models, and clinical samples. In summary, this dissertation advances the knowledge of RNA-ZnF molecular recognition and introduces technology for profiling RBP-RNA interactions in situ, addressing the challenges in understanding RNA-relevant diseases and therapeutics developments.