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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NeuroASPECT (Neuronal Alternative Splicing and RNA-Editing Crosstalk)

Teaser

Summary

One of the most intriguing questions in biology is to understand how in multicellular organisms the same genomic information can give rise to different cell types and tissues with distinct properties and functions. In particular, the mammalian central nervous system is a complex network of cells that relies on a tight regulation of gene expression that involves the coordination of post-transcriptional RNA mechanisms, such as alternative splicing (AS) and RNA-editing to boost genomic information and increase the repertoire of transcripts. Despite that RNA processing pathways present their highest prevalence in brain an integral view of the extent, evolutionary conservation and functional impact of neural regulated events is still missing.
Our project can be divided in three main parts, each of those targeting three objectives.
Generation of an extensive catalogue of tissue-specific alternative splicing events and RNA-editing sites, with special emphasis of those derived from neural tissues, of fifteen bilaterian species spanning more than 500 million years of evolution.
Assess the evolutionary conservation of neural regulated events across all the species, highlighting those that are conserved between mammals and other species and therefore suggesting a functional role for these events in the physiology of the mammalian nervous system.
Test the biological impact of highly conserved neural regulated events in the development and/or physiology of the nervous system.
Overall, our work resulted in valuable conclusions.
First, by making use of our extensive catalogue of tissue-specific AS events of fifteen species, we have found that AS is a prevalent molecular tool used by the nervous system of bilateria, suggesting an ancestral role of this post-transcriptional mechanism to increase transcript diversity in neurons. Moreover, we found that the AS events from neural samples present higher evolutionary conservation than other tissues tested, with some of them even preceding the chordate ancestor and implying a functional and ancestral role of these AS events in neurons development and function.
Finally, by testing some of these neural conserved AS events in the model system Drosophila melanogaster, we determined that defects in the inclusion of these events in the nervous system of the fly can result in severe neurological defects.
Therefore, we expect that our comprehensive catalogue of tissue specific AS events in conjunction with their evolutionary conservation provide a robust source for the scientific community to study the role of tissue regulated AS in normal conditions and disease in the nearby future.

Work performed

In order to generate the catalogue of tissue-specific alternative splicing events and RNA-editing sites, we have collected sequencing samples for public and in-house tissue-specific RNA of fifteen bilaterian species, that includes four mammals: human, mouse, cow and opossum; four non-mammalian vertebrates: chicken, frog, zebrafish and elephant shark and seven non-vertebrate species: amphioxus, sea urchin, centipede, fruitfly, mayfly, honey bee and octopus of eight major tissue groups: Neural, Muscle, Epithelial, Digestive tract, Kidney, Adipose, Ovary and Testis. All the gene and exon quantifications were performed using vast-tools (https://github.com/vastgroup/vast-tools) and for detection of RNA-editing sites we have made use of SAILOR (https://github.com/YeoLab/sailor). In average, we found that in average seven percent of the exons analyzed in all species are alternatively spliced, with human presenting the highest amount of AS exons. In terms of tissue-specificity, we obtained that for the majority of the species tested, the neural, testis and muscle tissue groups present the highest number of tissue-specific exons exons. All the events obtained from this analysis will be accessible soon at VastDB (http://vastdb.crg.eu/), were the information for human, mouse and chicken is already publicly available.
To evaluate the evolutionary conservation of tissue-specific exons, we have developed a computational pipeline to determine exon orthology of any species with genomic information available. Currently, we are working to adapt this pipeline also for the identification of conserved RNA-editing sites. Making use of out pipeline to the aforementioned species resulted in about 80% of the exons to be assigned as conserved between at least one pair of species. Moreover, for human tissue-specific exons, we found that 52% of the neural-specific exons and 46% of the muscle-specific exons are conserved among all vertebrates analyzed. In particular, the set of human highly conserved neural regulated events found in this project denote a valuable resource for future experimentation to evaluate the role of these post-transcriptional events in neural physiology and neurological diseases.
Finally, we have selected a couple of highly conserved neural AS events to test their role in the nervous system of the fruitfly Drosophila melanogaster. We found that deletion of these AS events in the nervous system results in flies with severe neurological defects, supporting the functions of these events in the proper physiology of neurons.

Final results

Our project has resulted in two major milestones for the scientific community. The first one is a catalogue of alternative splicing events and RNA-editing sites in fifteen bilaterian species. This, so far, represents till now the most extensive collection of AS events/RNA-editing sites in different tissues at a large phylogenetic scale. This will allow to any scientist interested in particular AS events and/or RNA-editing sites to easily evaluate their regulation in different tissues and their evolutionary conservation, thus expediting further experimentations. All this information will be soon accessible at VastDB (http://vastdb.crg.eu/), where the AS events for human, mouse and chicken are already available.
A second achievement of our proposal is the development and successful application of a pipeline to estimate exon orthology by using only genomic information. This tool represents to our knowledge the first of its kind and will simplify for the scientific community the assessment of evolutionary conservation at the exon level. Furthermore, we are working to expand our pipeline to also evaluate the evolutionary conservation of RNA-editing sites. The resulting software will soon be release in GitHub (https://github.com/) so that scientists can freely and easily apply our tool to any species of their interest.
We expect that our findings together with the resources generated in the course of the project will contribute and facilitate the design of new experiments that test the role of post-transcriptional events in any tissue and species and their influence to known diseases when their regulation is affected.