Cell type-specific alternative splicing (AS) of pre-mRNA regulated by RNA-binding proteins (RBPs) is widespread, but particularly prominent in the brain, driving gene isoform differences between a diverse range of neuron types. While several AS programs have been shown to be critical to the function of particular neuron types, previous studies have usually been limited to one or a few RBPs and cell types, resulting in a piecemeal understanding of these regulatory patterns. Towards a comprehensive view of the neuron type-specific AS regulatory landscape, we apply current computational and experimental methods to survey neuronal AS, infer its regulation by hundreds of RBPs, and experimentally validate regulatory predictions.
In Chapter 1, we examine AS in 133 transcriptomic cell types of mouse cortical neurons defined by single-cell RNA sequencing (scRNA-seq) and define neuron type-specific exons and some of their likely regulators. In Chapter 2, we leverage the rich transcriptomic dynamics of the cortical neuron dataset to systematically infer splicing regulatory network and predict RBP activity on the cell type level. We use the information theory-based method ARACNe to reverse engineer RBP-target regulatory networks and VIPER to infer differential RBP activity across neuron types in a workflow we call Master Regulator analysis of Alternative Splicing (MR-AS). RBP regulons predicted by MR-AS are consistent with high-confidence lists of RBP targets and are supported by motif and CLIP read distribution analyses. Estimation of cell type-specific RBP activity using the predicted regulons shows the expected decreases in RBP KO samples.
Chapter 3 focuses on two neuron type-specific AS regulatory programs as case studies, which we validate in vitro using embryonic stem cell (ESC)-derived neuron types. Elavl2 was predicted to drive neurons towards an MGE interneuron-specific AS profile. Elavl2 knockout in ESC-derived MGE interneurons causes modulation of exon inclusion consistent with the predicted regulation of MGE interneuron AS, shifting their splicing profiles towards those of CGE interneurons. We also identified a module of exons that show consistent AS between long- and short-projection neurons across multiple neuronal classes, which are shifted in the expected direction when ESC-derived interneurons are transcriptionally reprogrammed to reflect a long-axon globus pallidus-like neuronal identity.
In Chapter 4, we use the RBP regulons to predict RBP activity on a single-cell level and examine its variability, leading us to identify both neuron type-specific AS programs and a neuron type-orthogonal gradient of activity (NTOG). Exons associated with responses to neuronal depolarization and long-term potentiation show a gradient of inclusion across the NTOG, suggesting it may reflect differential activation of activity-dependent AS programs of the assayed neurons. Together, the results described in this thesis demonstrate the validity and broad utility of the inferred AS regulatory networks as a resource for elucidating RBP splicing regulation differences and their functional impact across neuron types.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/v453-g958 |
| Date | January 2023 |
| Creators | Moakley, Daniel |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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