Deciphering the role of RNA editing in zebrafish development
RNA editing was described for the first time in the 1980s, so it isn’t a new discovery. Since then, RNA editing was characterised in numerous organisms and it has been reported to function in an array of biological processes. RNA editing affects physiology and behaviour of animals from insect to human, by altering both, the sequence and structure of nervous system components. The most interesting roles of RNA editing described so far are: determination of castes in ants, adaptation to cold in octopuses. Most of all, RNA editing is crucial for correct development of the brain and nervous system of animals.
In addition, RNA editing was proposed to protect human (and other primates) genome against the expansions of Alu elements. Alu elements are repetitive sequences that are very abundant in our genome. What is more, Alu elements are believed to destabilise the genome by copying themselves across the chromosomes through the process of retrotransposition (transposition involving RNA). ADAR, one of the enzymes responsible for RNA editing, was found to bind Alu transcripts, edit their sequence and therefore block subsequent transposition to new genomic locations.
Besides extensive research and multiple proposed functions in various organisms, there is still no consensus for the purpose of RNA editing. We would like to study the role of RNA editing in developing embryo. Obviously, we cannot conduct this study in human, therefore we will use zebrafish (Danio rerio), as it is easy to maintain in the lab and gives access to very early developmental stages, while being relatively close to human (human share most of the genes with zebrafish).
We’ll to characterise RNA editing in zebrafish, by sequencing parental genomes and the transcriptomes of developing embryos at several stages of development. As RNA editing is expected to create difference between transcripts and genome sequence, subsequent comparison of transcripts with genome sequence will allow genome-wide detection of RNA editing. This will allow us not only to create most comprehensive RNA editing catalogue of developing embryo, but also to identify the changes in RNA editing throughout embryo development.
Summary of obtained results
During this project, I’ve developed novel bioinformatics methods to map RNA editing sites in the transcripts using data generated from next-generation sequencing (NGS) experiments. This method allowed us to identify transcripts edited by ADAR proteins. Many serve as key regulators of early development, responsible, for instance, for the normal definition of the anterior-posterior axis of the body. Interestingly, when we disturbed this protein in embryos, we obtained larvae with heavily affected body axis. Finally, we have created zebrafish mutant lacking Adar protein, that will allow further studies of this process. A detailed investigation into the role of RNA editing in early vertebrate development will contribute to a better understanding of gene expression regulation during embryonic growth.
I have been working on my research at the Laboratory of Zebrafish Developmental Genomics, lead by Dr Cecilia Winata at the International Institute of Molecular and Cell Biology in Warsaw. Apart from giving me access to cutting-edge technologies in molecular biology and promoting my personal growth, the POLONEZ programme has provided me with great opportunity to cooperate with researchers from all around the globe.
- Animal handling training according to Polish laws (PolLASA)
- Identification of previously unannotated rRNA clusters in zebrafish genome
- Protocol optimisation for rRNA depletion for Eukaryotes and Bacteria using TEX
- Established protocols for direct RNA sequencing using Oxford Nanopore sequencer
- Establishment of protocolos for Adar knock-down and overexpression
- RNA-seq of control, Adar knock-down and overexpression at 2 hpf (maternal) and 5.3 hpf (zygotic transcripts)
- Identification of Adar targets in maternal and zygotic transcrtipts
- Establishment of novel transgenic line (Adar KO)
- Rescue experiments with human Adar ortholog and enzymatically inactive Adar
- Phenotypic characterisation of Adar knock-down and knock-out lines
- Development of novel tool for accurate RNA editing detection from RNA-seq (REDiscover)
- Adaptation of REDiscover to detect multiple types of RNA modifications from RNA-seq
- Polish Illumina User Symposium, Poznań, Poland (11-12 Oct 2018) - invited lecture: “NGS tips & tricks”
- European Epitranscriptomics Network meeting, Malaga, Spain (8-11 Oct 2018) - invited seminar: “Toward new methods for detection of RNA modifications using second and third generation sequencing”
- #NGSchool2018 Summer School, Lublin, Poland (16-23 Sep 2018) - lecture: “MinION explained: principles, running & hacking it.”
- 6th Central European Summer Course on Mycology, Szeged, Hungary (6-11 Jul 2018) – invited lecture: “Evolutionary innovations as a result of inter-species genome hybridisation.”
- Genome Bioinformatics for Health, Großbothen, Germany (25-27 Jun 2018) – invited seminar: “NGS in Biomedicine: Are we ready for SMRT revolution?”
- EMBL Conference: The Epitranscriptome, Heidelberg, Germany (25-27 Apr 2018) – poster: “RNA editing in embryonic development”
- 10th European Zebrafish Meeting 2017, Budapest, Hungary (3-7 Jul 2017) - poster: “Deciphering the role of RNA editing in vertebrate development”
Organisation of scientific events
- #NGSchool2017: Single-cell sequencing, Jachranka, Poland (10-17 Sep 2017) – main organiser
- RNA Club meets #NGSchool, IIMCB, Warsaw (series of NGS-related workshops, Feb-May 2018) – main organiser
- Nuclear landscapes, CeNT UW, Poland (14-16 Sep 2018) – one of main organisers, member of scientific committee
- #NGSchool2018: Nanopore sequencing & personalised medicine, Medical University Lublin, Poland (16-23 Sep 2018) – main organiser
- DoScience!, Warsaw, Poland (series of seminars) – invited & hosted numerous international speakers
Detailed information about the project
- Project Duration: 01.01.2017-31.12.2018
- Project number: 2015/19/P/NZ2/03655
- Project title: “Deciphering the role of RNA editing in zebrafish development”
- Project leader: Dr. Leszek Piotr Pryszcz
- Source of funding: National Science Centre, Poland, POLONEZ
- Budget: 921 064 PLN
This project has received funding in National Science Centre (Poland) Polonez-1 framework from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 665778.