PROJECT SUMMARY
Alternative RNA splicing enables generation of different spliced mRNA isoforms that can encode functionally
distinct proteins. Splicing factors (SFs) are RNA-binding proteins that regulate splicing in a dose-dependent
manner and are frequently dysregulated in diseases. Serine/arginine-rich (SR) proteins (SRSF1 to 12, and SR-
like members TRA2a, TRA2ß) are a family of essential SFs causatively implicated in a wide range of human
pathologies. Elucidating how SR proteins are regulated is crucial to advance our understanding of the
fundamental processes that control gene expression in eukaryotes and to target diseases with SF defects. Here,
we will focus on post-transcriptional regulation as an important modulator of SR protein expression, and a
potentially actionable pathway for tool and therapeutics development. SR protein genes contain ultra-conserved
non-coding exons, called poison-exons (PEs), which control SR protein auto-regulation. Conservation of PE
sequences across species suggests their importance in regulating SFs. However, how PEs regulate gene
expression and maintain broader SF homeostasis, and how they contribute to fundamental cell functions remain
poorly understood. We hypothesize that PEs play a critical role in maintaining a tight regulation of SF levels,
which is necessary for normal cell functions. Aim 1 will define the mechanisms of SR protein regulation and
cross-regulation via PEs using splicing reporter minigenes, a CRISPR/Cas9 library targeting SFs, and long-read
RNA sequencing. By identifying the SR proteins and SFs that are interconnected and co-regulated through PE
splicing, these findings will provide a comprehensive map of the SR protein regulatory network and will uncover
novel principles of post-transcriptional gene regulation. Aim 2 will define the functional role of SR protein PEs in
development and cell differentiation using CRISPR/Cas9 to delete PE sequences in vivo in mouse embryos and
in vitro in human cell differentiation models. These findings will reveal cell types and cellular states that require
PEs to function normally, as well as PE targets in vivo and in vitro. Aim 3 will develop approaches to modulate
PE splicing and SR protein levels. These approaches will be used to infer SR protein binding rules, and to probe
PE function in relevant disease models. The proposed aims leverage our lab’s expertise in developing tools and
models to study splicing dysregulation in diseases. Completion of these aims will identify novel molecular
mechanisms by which PEs regulate SF homeostasis and cellular functions, and provide new tools to manipulate
SR protein levels. The regulatory mechanisms uncovered here are likely to have broad relevance to many SFs,
the majority of which contain PEs. Manipulating SF levels by targeting PEs could lead to therapeutic approaches
for diseases with SF defects, such as neurological disorders, cardiac myopathies, diabetes, lupus, or cancer.