RNA Partitioning Into Nuclear Speckles: From Spatial Organization to Cooperative Kinetics

Biomolecular condensates formed by liquid–liquid phase separation provide a physical mechanism for organizing intracellular biochemistry. A central open question is how molecularlevel information, such as RNA sequence, is translated into spatial partitioning and how this spatial organization in turn regulates biochemical reactions. In this dissertation, we address this question using nuclear speckles, nuclear condensates enriched in splicing factors, as a model system to establish a quantitative link between RNA sequence, phase partitioning, and RNA splicing.

In the first part, we establish that RNA sequence encodes its propensity to partition into nuclear speckles. Using synthetic sequence design and quantitative imaging, we show that sequence features containing hallmarks of pre-mRNA dictate nuclear speckle partitioning. These features drive speckle partitioning through interactions with spliceosomal components and splicing factors, thereby establishing nuclear speckle partitioning as a sequence-encoded property arising from RNA–protein interactions.

In the second part, we demonstrate that nuclear speckle partitioning functionally regulates pre-mRNA splicing. Using high-throughput imaging measurements, we show that speckle enrichment correlates with enhanced splicing kinetics and switch-like transition in exon inclusion. Quantitative models that account for these observations highlight the roles of cooperative RNA–protein interactions and spatial organization in regulating splicing kinetics and decisions.

In the final part, we show that RNA exhibits non-uniform spatial organization within nuclear speckles. Super-resolution imaging combined with a minimal physical model reveals that competing interactions can drive distinct radial distributions of RNA within the condensate, providing an additional layer of spatial regulation beyond phase partitioning.

Together, these results support a physical framework in which RNA sequence encodes effective interactions that determine nuclear speckle partitioning; and speckle partitioning is further coupled to splicing outcomes. This work establishes nuclear speckles as a quantitative model system for studying the coupling between phase separation and biochemical reactions and provides a general framework for understanding how spatial organization in living systems emerges from molecular interactions and shapes functional outcomes.