Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - G-EDIT (Mechanisms of RNA-guided genome editing in eukaryotes)
Teaser
The goal of this project is to contribute to our understanding of RNA-mediated epigenetic mechanisms of genome regulation in eukaryotes. Ciliated protozoa offer a fantastic opportunity to investigate the complex process of trans-generational programming of chromosomal...
Summary
The goal of this project is to contribute to our understanding of RNA-mediated epigenetic mechanisms of genome regulation in eukaryotes. Ciliated protozoa offer a fantastic opportunity to investigate the complex process of trans-generational programming of chromosomal rearrangements, which is thought to serve as a form of immune defense against invasive DNA. Developmental processes in ciliates include extensive rearrangements of the germline DNA, including elimination of transposons and the precise excision of numerous single-copy elements derived from transposons. This process is considered to be maternally controlled because the maternal genome provides essential information in the form of RNA that determines the offspring\'s genome content and organization. This programmed DNA subtraction, the so-called ‘RNA scanning’ process, is mediated by trans-generational comparison between the germline and the maternal somatic genome. One of the most intriguing questions is how a complex population of small RNAs representing the entire germline genome can be compared to the entire rearranged maternal genome, resulting in the efficient selection of germline-specific RNAs, which are able to target DNA deletions in the developing genome. All this occurs in a very short time and involves a massively coordinated transport of all the components between three types of nuclei. This project focuses on characterizing the molecular machinery that can orchestrate the massive genome rearrangements in ciliates through nucleic acids and protein interactions. It also addresses the question how RNA targets DNA cleavage at the right place. In addition, this project aims to investigate the role of RNA in guiding chromosomal rearrangements in other eukaryotic systems, particularly in human cancer cells where genome editing often occurs on a large scale. This work may be the first step in providing novel insights into the process of programmed DNA rearrangements in higher eukaryotes.
The main goals:
1. Characterizing Dicer-like proteins involved in Paramecium development
2. Roles of Piwi proteins in Paramecium development
3. Testing the requirements for DNA cleavage targeting by small RNAs
4. Role of chromatin in Paramecium development.
5. Investigating RNA-templated chromosomal rearrangements in human cells.
Work performed
Aim 1. My team has performed an in-depth characterization of development-specific Dicer proteins with special attention to their cleavage preferences. We discovered that Paramecium Dicers cleave dsRNA with a strong bias towards 5’ and 3’ end sequence motifs, which allows the enzymes to produce small RNAs that map preferentially to the ends of transposon remnants (called IESs), which in turns allows excision of these elements from the genome. This work has been published in Cell (Hoehener et al, Cell. 2018 Mar 22;173(1):234-247.e7. doi: 10.1016/j.cell.2018.02.029).
Aim 2. We are currently in process of characterizing developmental-specific Piwi proteins in Paramecium. We performed knockdown and RNA-coimmunoprecipitation which allowed us to discover that one of the proteins binds sRNAs that originate from single stranded precursors. This is in contrast to other Paramecium Piwis which bind sRNAs produced by Dicer cleavage. This would be the first example of a Piwi pathway existing in non-animal model. Based on the small RNA mapping we assume that the Piwi protein may be involved in transposon silencing, however, a lot of the small RNAs map to non-transposon genomic loci.
Aim 3. We have also shown that after providing paramecium cell with a piece of DNA that resembles an excised IES, the cell utilizes it to excise the corresponding region from the chromosome. This not only helped to understand the mechanism of developmental DNA elimination but also provided us with a useful tool for somatic gene knockout. We can target this way any sequence flanked by TA nucleotides which leads to a deletion. This experiment was included in another Cell paper: Allen et al., Cell. 2017 Mar 9;168(6):990-999.e7. doi: 10.1016/j.cell.2017.02.020.
Aim 4. We have been investigating the role of chromatin in developmental genome rearrangement. Based on previous research on Tetrahymena it is assumed that IESs and transposons need to be heterochromatinized prior elimination. We have discovered that in Paramecium the mechanism is completely different. We found a chromatin remodeler ISWI which is required for DNA elimination and its role is to move nucleosomes away from the target regions making them nucleosome free. This is needed to provide access for the excision machinery. The targeting of ISWI to the correct loci is mediated by small RNAs. I believe this is a first example of RNA-mediated euchromatinization. This work is currently in preparation for submission. We also published paper about evidence of absence of cytosine methylation in Paramecium which is important to a wide group of researchers who rely on antibody-based evidence. We showed that antibodies may lead to false-positive results and it is therefore important to use several alternative approaches, such as bisulfite sequencing and mass spectrometry (Singh et al., PLoS One. 2018 Oct 31;13(10):e0206667. doi: 10.1371/journal.pone.0206667)
Aim 5. Investigating RNA-templated chromosomal rearrangements in human cancer cells is still at an early stage and we do not have any positive results yet.
Final results
I expect that the proposed research will allow us to understand the mechanism of RNA-mediated DNA elimination from several perspectives, such as RNA molecules, chromatin and protein machinery. It may also lead to the use of the knowledge to edit the genomes in other model organisms such as human.