PROPOSAL SUMMARY/ABSTRACT Alternative splicing of pre-mRNA plays a key role in the regulation of gene expression and contributes to protein diversity between tissue types. Recent work has demonstrated that tissue-specific “microexons” that are 3-27 nucleotides in length are highly conserved in metazoans, play a prominent role in neuronal development, and are frequently mis-spliced in patients with autism spectrum disorder. Work from the last decade has demonstrated that splicing frequently occurs during transcription elongation (co-transcriptionally), with introns rapidly excised following their synthesis. It is unclear how microexons can be defined rapidly enough to be spliced during transcription elongation when both the 3' and 5' splice sites emerge from the RNA Polymerase II (Pol II) exit channel nearly simultaneously. Additionally, the co-dependence of microexon splicing with downstream mRNA processing events is unknown. The work outlined in this proposal, broken down into three independent aims, will fill substantial gaps in our understanding of neuronal microexon splicing regulation. In Aim 1, I will adapt sequencing-based methods pioneered in the Neugebauer lab to investigate the relationship between microexon splicing and transcription elongation. I hypothesize that microexons are spliced under different transcriptional parameters than “conventional” exons which are on average ten times longer. In Aim 2, I will analyze single molecules of mRNA to determine processing steps that co-occur in mRNAs with microexons. I anticipate that microexons will serve to influence downstream processing events (e.g polyA cleavage site choice). Finally, in Aim 3, I will use a series of splicing reporters to investigate the influence of local mRNA sequence to microexon splicing. This proposal addresses many unresolved questions related to neuronal microexon regulation.