Final Report Summary - NRX CODE (Deciphering the neurexin code in neuronal circuitry)
Nrx are important synapse organizing molecules and are known to play a pivotal role in synapse formation. Through alternative splicing of transcripts derived from three Nrxn genes thousands of isoforms can be potentially generated, making these proteins promising candidates for determination of some aspect of neuronal identity, wiring specificity and/or synaptic properties. Mutations in Nrxn genes were demonstrated to be involved in many neuropsychiatric disorders such as schizophrenia, autism, and addiction. Thus, the understanding of the functional role of Nrx is a valuable research topic not only for gaining our elucidation of basic mechanisms of the formation and the organization of the CNS, but also an important issue in our understanding of neuropsychiatric disorders.
Despite the importance the role and real extend of Nrx diversification through alternative splicing is not known. In this project we attempt the elucidation of neurexin diversity at mRNA and protein level, using new technological developments; third-generation sequencing and selected-reaction monitoring (SRM) mass spectrometry.
Using third-generation sequencing technology we performed for the first time in depth sequencing of alpha-Nrxn mRNA transcripts. We could demonstrate the true diversity of Nrxn transcripts in the adult mouse brain. The obtained data provides inside into the some rules of combinatorial alternative splicing of alpha-Nrxn. We demonstrate that the alpha-Nrxn repertoire is dominated by certain isoforms, the repertoire correlates with cellular diversity, and does not reach the theoretically predicted diversity. This was in particular true for Nrxn3. We could demonstrate that the low number of Nrxn3 variants results from strong coupling between certain splicing events. Analysis of some invariant splicing events at alternative splice segment 5 (AS5) of Nrxn3 led us to identification of new regulatory elements in the 3’-untranslated region (3’UTR) in certain Nrxn3 variants.
During this project we successfully established and applied SRM mass spectrometry assays for identification and quantification of different Nrx proteins. Using this approach we could confirm the existence of Nrx variants containing a new alternatively spliced exon in Nrx1 and Nrx3, which we identified during this project. Using this assays we could for the first time demonstrate relative and absolute contribution of different Nrx variants to the pool of synaptic proteins, and in different multi-protein complexes.
Additionally, during this project we created and optimized for in vivo applications a splice reporter system which allows us monitoring of splice events with the single cell resolution. Using this system we could demonstrate that substantial differences in splicing of certain alternatively spliced segments in Nrxn genes occur not only in different cell populations but also in individual cells of same cell population.
Despite the importance the role and real extend of Nrx diversification through alternative splicing is not known. In this project we attempt the elucidation of neurexin diversity at mRNA and protein level, using new technological developments; third-generation sequencing and selected-reaction monitoring (SRM) mass spectrometry.
Using third-generation sequencing technology we performed for the first time in depth sequencing of alpha-Nrxn mRNA transcripts. We could demonstrate the true diversity of Nrxn transcripts in the adult mouse brain. The obtained data provides inside into the some rules of combinatorial alternative splicing of alpha-Nrxn. We demonstrate that the alpha-Nrxn repertoire is dominated by certain isoforms, the repertoire correlates with cellular diversity, and does not reach the theoretically predicted diversity. This was in particular true for Nrxn3. We could demonstrate that the low number of Nrxn3 variants results from strong coupling between certain splicing events. Analysis of some invariant splicing events at alternative splice segment 5 (AS5) of Nrxn3 led us to identification of new regulatory elements in the 3’-untranslated region (3’UTR) in certain Nrxn3 variants.
During this project we successfully established and applied SRM mass spectrometry assays for identification and quantification of different Nrx proteins. Using this approach we could confirm the existence of Nrx variants containing a new alternatively spliced exon in Nrx1 and Nrx3, which we identified during this project. Using this assays we could for the first time demonstrate relative and absolute contribution of different Nrx variants to the pool of synaptic proteins, and in different multi-protein complexes.
Additionally, during this project we created and optimized for in vivo applications a splice reporter system which allows us monitoring of splice events with the single cell resolution. Using this system we could demonstrate that substantial differences in splicing of certain alternatively spliced segments in Nrxn genes occur not only in different cell populations but also in individual cells of same cell population.