Alternative Splicing Control of Trim46 Regulates Formation of the Axon Initial Segment

Alternative splicing produces mRNA isoforms of distinct structure and function from the same gene. Neuronal polarization has been shown to be regulated by alternative splicing in the brain. Loss of neuronal enriched splicing factor PTBP2 causes deleterious effects in neuronal maturation and regulates genes involved in axon formation. We identify microtubule associated protein involved in axon formation, Trim46 as a PTBP2-specific target through multi-pronged approaches. We further show that non-sense mediated mRNA decay pathway (NMD) degrades transcripts containing premature termination codons (PTCs) of Trim46 isoform to control its steady-state expression. We used gene editing tool CRISPR/Cas9 to ablate alternative splicing and NMD control of Trim46 in mouse embryonic stem cells (mESC). Differentiation of mutant mESCS into neurons show defects of AnkG localization in the axon initial segment, demonstrating the essential roles of alternative splicing and NMD control during axon formation. We further performed gain and loss of function analysis to show that the long and short TRIM46 isoforms affect microtubule stability differently. In summary, alternative splicing and NMD control of Trim46 isoforms regulates formation of the axon initial segment.