Alternative splicing (AS) of human genes is pervasive and greatly expands the repertoire of protein and RNA products arising from the human genome. AS is critical for cellular differentiation and identity, and its dysregulation has been causally linked with a broad and expanding array of human diseases, including muscular dystrophies, neurodegenerative disorders and cancers. However, we currently have limited insight into the regulation of AS at both the local (gene) and global (genome-wide) levels, due to a lack of tools that provide direct, high-resolution, and quantitative views into the splicing process. This deficit has in turn roadblocked progress in understanding how splicing is regulated to confer cellular identity and to control differentiation processes. The eight introns per average human gene are processed co-transcriptionally through spliceosomal subunits and regulatory factors binding to specific sequences in nascent RNA. These cis-elements are typically within introns and thus act only from when they emerge from RNA polymerase to when they are spliced out. Consequently, in order to dissect splicing regulation mechanisms, we need to determine how fast splicing occurs and the order of intron excision across nascent transcripts. We recently developed nanopore analysis of CO- transcriptional Processing (nano-COP) that measures the kinetics, order and coordination of splicing of endogenous genes in vivo. Nascent RNA is purified and then directly sequenced using the Oxford Nanopore platform to obtain long reads. We found that splicing kinetics is influenced by intron length and proximity to alternatively spliced exons, that splicing order does not follow the order of transcription and that neighboring introns have the propensity to be spliced coordinately at the same time. The goal of this grant is to determine how cis-acting elements and trans-acting factors impact human splicing kinetics, splicing order and splicing coordination.
Specific Aim 1 : Determine how trans-acting factors impact splicing dynamics. We will study eight RNA-binding proteins that are connected to splicing regulation by our analysis or other studies. To diminish secondary effects, we will use an inducible degradation system to degrade target factors within hours. We will perform subRNA-seq and nano-COP to study splicing dynamics after the loss of each factor.
Specific Aim 2 : Determine the role of cis-acting elements in dictating splicing dynamics. We will determine how changes to splice site sequences and other cis-elements alter splicing kinetics and alternative splicing. We will use CRISPR-Cas9 and leverage natural genetic variants to study perturbations to cis-elements.
Specific Aim 3 : Determine the relationship between splicing dynamics and AS during human myogenesis. We hypothesize that key trans-acting factors control splicing kinetics that in turn affect AS. We will study how splicing dynamics change during myogenesis using nano-COP. The roles of myogenesis splicing regulators in controlling splicing dynamics will also be investigated. In sum, changes in splicing kinetics will be associated with AS outcomes to determine models of how splicing is regulated by splicing dynamics.
The proposed research is relevant to public health, because dysregulated alternative splicing leads to a broad range of diseases, including many cancers and neurological disorders. Our proposed research will shed light on how splicing is regulated, which could reveal novel therapeutic strategies to rescue disease-causing splicing defects. As such, the proposed research is relevant to the part of the NIH's mission that seeks to develop fundamental knowledge to inform our diagnosis and treatment of human disease.
