Alternative splicing (AS) is a major mechanism that generates the vast transcriptome and proteome diversity
from the limited genome. Spatial and temporal regulation of AS contributes to cell differentiation and lineage
determination. Mutations that disrupt splicing cause a wide range of diseases including neurodegeneration,
muscular dystrophies, and cancer. Therefore, understanding and targeting splicing is essential to the future of
precision medicine. In the past 3 decades, AS studies have focused on cis elements and their associated trans
factors, such as RNA binding proteins (RBPs). The laws of thermodynamics dictate that RNAs fold into low free
energy structures in the context of RNA-protein complexes (RNPs), however, very little is known about how
pre-mRNA structures in large RNPs control splicing, and various RNA processing events in general. Our lab
invented several chemical crosslink-ligation based methods that enabled direct analysis of transcriptome-wide
protein-independent RNA 2D and 3D structures in vivo (PARIS and SHARC). In this proposal, in Aim 1, we
develop and benchmark a high throughput technology, SHARCLIP, to directly capture all RNA structures,
RNA-RNA, RNA-protein, and protein-protein interactions together. SHARCLIP is conceptually similar to
methods that analyze protein-mediated chromatin conformations, e.g., ChIA-PET and hiChIP, and will bring
1D RNP interaction studies to higher dimensions. In aim 2, applying PARIS and SHARC to chromatin
associated RNAs, and the new method SHARCLIP to key splicing regulators, we will build transcriptome-wide
RNA-structure and RNA-protein interaction models and deconvolve their dynamics in 2 ENCODE cell lines
HepG2 and K562, as well as 6 brain cell lineages from a human iPS differentiation system. In aim 3, further
integrating cell type specific AS programs and disease variants implicated in splicing, we will test whether
mutations act by altering the structures. With the structure models as a guide, we will use a combination of
CRISPR genome editing and structure-perturbing antisense oligos to test the roles of specific structures in
regulating splicing in neuronal differentiation. Together, this proposal will produce a new technology that
simultaneously capture multi-valent RNA-protein interactome and RNA structurome in vivo, establish a
structure-based splicing code, provide an important resource to enable mechanistic studies of RNA processing,
and pave the way for future therapeutic targeting of splicing.