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Functions and evolutionary impact of transcriptomic novelties in the vertebrate brain

Periodic Reporting for period 4 - NEURAL AS (Functions and evolutionary impact of transcriptomic novelties in the vertebrate brain)

Reporting period: 2019-10-01 to 2020-12-31

The aim of this project was to investigate the functional impact of a specific type of genomic novelty (neuronal-specific microexons) in the development and function of vertebrate brains. For this, we needed to develop tools to identify and characterize such microexons (Aim 1): tiny parts of genes that are included in the proteins only in the neuronal cells, thus creating an alternative protein isoform in this cell type that has a distinct molecular function that contributes to differentiate it from the rest of cells in the body. Once identified, we investigated their function in vertebrate models (Aim 2). For this, we deleted them from the genomes of zebrafish and mouse cells, and measured the functional impact this has on the differentiation of neurons and the development and function of the central nervous system. Finally, we wanted to understand how these functions are played at the molecular level (Aim 3). For that, we hypothesized that in many cases these microexons will be modulating protein-protein interactions in a neuronal-specific manner.

We succeeded in identifying VN-AS exons as well as other neural (micro)exons with different evolutionary ages. Our functional characterization in vivo has shown that, while most of these exons are not essential on their own in lab conditions, they subtly affect different characteristics at different organizational levels. Finally, the resources and tools developed in this project have allowed us to contribute to many other research projects. These include further insight into the role of microexons and other alternative exons in autistic spectrum disorders and Huntington's disease, making this project more appealing from a biomedical perspective.
During the course of the project, we have identified and characterized the exons and microexons of interest: those that are only present in vertebrates but not in any invertebrate animal, and that are neuronal-specific across vertebrates (what we call Vertebrate- Neural- Alternatively Spliced exons, or VN-AS exons). We have also characterized their regulation across tissues and neuronal differentiation time courses, and made a public database with all this information (VastDB; vastdb.crg.eu). To achieve this goal, we have also developed the ExOrthist, a tool to infer exon homology relationships.

We have also implemented the necessary methodology to selectively delete microexons in zebrafish, and generated 21 lines with microexon deletions, 18 of which corresponded to clean deletions with no effect on overall gene expression. We have assessed phenotypes in these lines at different organizational levels using different tests. These range from study of neuritogenesis in vivo and in culture, transcriptomic changes using whole-embryo RNA-seq, basic locomotion and sensory tests using Daniovision, and social behaviour assays with a custom set-up. We have identified several microexons with defects on neuritogenesis and/or social behavior, associated with specific transcriptomic patterns. For a few cases, we could infer and test the molecular functions for these VN-AS microexons.

Moreover, these analyses led to unexpected evolutionary findings, which we have followed up in different publications (e.g. Torres-Mendez et al 2019 and 2021, Marletaz et al 2018). In particular, we found that the programs of neural microexons originated in bilaterian ancestors, much earlier than the origin of vertebrates. This means that, although vertebrate-specific neural microexons are a key component of the VN-AS program, the regulatory machinery originated before the origin of our group. Also, it means that other lineages (e.g. flies) have evolved their own programs of neural lineage-specific microexons, which we have also investigated.
This project is the largest study of alternative exon function in an in vivo system. Therefore, it will provide a more comprehensive overview of how different targets of a gene regulatory network contribute to the strong phenotype observed for the master regulator. Moreover, it has allowed the development of resources and software that were not available at the beginning of the action.
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