Regulation of synaptic adhesion molecules by RNA processing

The highly specialized neuronal cells and their complex connectivity pattern is the hallmark of central nervous system organization. Synapse formation and specification are crucial steps in generating this complex wiring and proper establishment of functional synapses is essential for correct brain function. Cell adhesion molecules are key mediators in synapse specificity and moreover in generating functional synapses. Their diversification by alternative mRNA splicing, which can amplify the gene pool, is proposed to underlie molecular codes for synapse formation (Gomez et al., 2021). Additionally, alternative splicing is thought to be a stronger driver for synapse diversification than differential gene expression, as determined by transcriptomic approaches (Furlanis et al., 2019). However, due to mechanisms influencing translation of transcripts on multiple levels, it is hard to predict the proteome only based on transcriptome studies. Moreover, the expression, localization and function of individual protein isoforms, or “proteoforms”, generated by alternative splicing remains enigmatic.
In this thesis, I combine multiple approaches to highlight the importance of probing individual proteoforms to study the generation of a molecular code underlying synapse specification. By combining targeted proteomics with the analysis of specific knock-in and knock-out mice, I could characterize a non-canonical proteoform of the synaptic adhesion molecule family of Neurexins. Alternative splicing and inclusion of an alternative exon at the splice segment 5 (AS5) of Neurexin 3 (Nrxn3) leads to specific expression of this proteoform in GABAergic interneurons and at pre-synaptic GABAergic terminals. Strikingly, this stands in contrast to the abundance of Nrxn3-AS5+ transcripts and could be determined to depend on translational repression elements in the 3’ untranslated region of Nrxn3. Aberrant splicing at AS5 and altered incorporation of repression elements leads to a drastic drop in NRXN3 protein levels while mRNA levels of Nrxn3 are unaltered.
Moreover, NRXN3 AS5+ protein isoforms change their membrane association from a transmembrane to a GPI-anchored form. Deletion of this novel GPI-anchored NRXN3 protein isoform leads to an impairment of synaptic transmission in a subset of CCK positive GABAergic synapses in the dentate gyrus.
Thus, my thesis supports the use of more refined proteomic approaches to study the pleiotropy of synaptic molecules shaping synapse diversity. Finally, and to promote and collect already validated and successfully used targeted mass-spectronomy assays, we develop a database (https://scheiffele-SYNCODE.scicore.unibas.ch/) to strengthen the efforts of studying synaptic function on the proteome level.