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NEURAL AS Report Summary

Project ID: 637591
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - NEURAL AS (Functions and evolutionary impact of transcriptomic novelties in the vertebrate brain)

Reporting period: 2015-04-01 to 2016-09-30

Summary of the context and overall objectives of the project

Not yet applicable

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

- Aim 1.1: the major objectives of this Aim have been achieved. All the proposed high-throughput RNA sequencing (RNA-Seq) has been performed and the databases of alternative splicing profiles for various species have been constructed as planned (Tasks 1 and 2). A public web resource (Task 3) has been built for vertebrates (manuscript on the analysis recently submitted for publication;, and is expected for invertebrates and other additional vertebrates by June 2017 (upon publication of the first resource). Moreover, due to novel discoveries on the conservation of neuronal microexons and their regulation across animals, we have expanded these Tasks to include further invertebrate species (Drosophila, C. elegans, Strigamia maritima, Octopus). With all these information, we have already identified dozens of VN-AS exons (as well as exons and microexons conserved between vertebrates and invertebrates)(Task 4) and these have been extensively validated by RT-PCRs (Task 5).
- Aim 1.2: we have developed and optimized a FACS-based protocol to isolate cells expressing fluorescent proteins from zebrafish embryos. We have currently used it to perform RNA-Seq data of HuC:GFP-positive cells and we will soon use it for Ngn:RFP (Task 6). In addition, we are developing the use of methods based on single-cell transcriptomics, which are expected to be more powerful (see below). Regarding Task 7, we have analysed multiple RNA-Seq datasets of mammalian differentiation that became available, and currently have good resources for in vivo and in vitro regulation of neural microexons in mammals.
Task 8: we have analysed RNA-Seq data for all mammalian KOs and KDs of the proposed splicing factors. In addition, we started the generation of zebrafish lines KO for Srrm4 and Srrm3 (the main regulators of neural microexons). We have recently obtained the first embryos from a F2 generation line carrying a mutation in Srrm4 that abolishes the start codon, and are currently studying its impact and validity. An important delay in this Task (and Task 12) has occurred due to a massive accident in the zebrafish facility at PRBB, in which 90% of the fish of the facility died due to a chemical contamination. This occurred in April 2014, and all our KO lines for Srrm3 and Srrm4 died, except for the one mentioned above. However, this Task was expected to be finished by month 30, so we do not see special issues to complete it at the current working pace.
Task 9,10: these tasks are currently not being implemented as planned, as we are developing a single-cell RNA-seq-based approach that will hopefully allow higher throughput and resolution. In addition, we have at our disposal a much larger resource of RNA-Seq data for different tissues and cell lines in multiple organisms than previously anticipated.
Aim 2: as mentioned in the proposal, we decided to focus on microexons due to their scientific novelty and interest. We have currently selected 23 microexons (and we expect to reach at least 30) (Task 11), and are currently at different stages of generating stable KO lines for all these exons (Task 12). Despite the major accident occurred in the fish facility, we have recently obtained our first homozygous KO line (for a microexon in Evi5b) and we have identified F1 heterozygous founders for 3 other microexons. Based on the current pace, we should be able to generate all the KO lines within the expected time frame. We have not yet observed any gross phenotype in the only homozygous line we generated, but we will start finer experiments soon. Thus, Task 13 has not been initiated (it was planned to be initiated after month 18). Finally, regarding Task 14 (generation of exon KOs in mammalian embryonic stem cells and differentiation to neurons), we have set up the workflow to obtain exon KOs (we have currently generated lines for 7 exons), but we have not started the assays of neuronal differentiation.
- Aim 3: we have mapped all alternative exons to available protein structures, and performed models for those that have not been experimentally determined. We have also identified exons likely involved in interactions, based on structural data or overlap with interaction motifs. However, such results have been rather disappointing so far. We will try docking approaches in collaboration with a local expert on protein-protein interactions (Dr. Aloy, IRB, Barcelona) to improve these results (Task 15). On the other hand, Task 16 and 17 will be performed mainly in collaboration with Dr. Blencowe (University of Toronto, Canada), who has set a high-throughput workflow for these analyses, and has kindly agreed to collaborate with us.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

not yet applicable
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