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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - NEURAL AS (Functions and evolutionary impact of transcriptomic novelties in the vertebrate brain)

Teaser

Summary

The aim of this project is 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 need to identify 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 need to investigate their function in vertebrate models (Aim 2). For this, we delete them from the genomes of zebrafish and mouse cells, and measure the functional impact this has on the differentiation of neurons and the development and function of the central nervous system. Finally, we want 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. If these Aims are accomplished we will better understand our brains, from a function and evolutionary level. Furthermore, neuronal microexons have been implicated in autistic spectrum disorders, making this project more appealing from a biomedical perspective.

Work performed

We have so far managed to identify the microexons of interest: those that are only present in vertebrates but not in any invertebrate animal, and that are neuronal-specific in all species (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 these information.
We have implemented the necessary methodology to selectively delete microexons from zebrafish, and generated 22 lines with microexon deletions. We are currently investigating the phenotypes with 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 tests. We have identified several microexons with defects on neuritogenesis with associate with specific transcriptomic patterns.
In addition, from the work implemented in Aim 1, we obtained a surprising finding: 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.

Final results

We expect to characterize all phenotypes from the molecular to the organismal level for over 20 neuronal microexons, and obtained high details for some of them. Then, we expect to be able to rescue these phenotypes only with the microexon-containing mRNAs, proving the importance of these microexons. With all these information on the function of microexons at multiple levels, we will propose hypothesis on how they could have influenced the origin of vertebrates, particularly on shaping our remarkably complex brains. It will also important to integrate these results with the phenotypes observed in the regulators of microexons Srrm3 and Srrm4. Finally, given the findings that the different bilaterian lineages have evolved their own programs of specific neural microexons, we have also started investigating the function of this program in fruit flies, with the aim of assessing whether the roles of VN-AS microexons are similar to those of fly-specific ones or unique of vertebrates.