Periodic Reporting for period 4 - G-EDIT (Mechanisms of RNA-guided genome editing in eukaryotes)
Okres sprawozdawczy: 2020-11-01 do 2021-04-30
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.
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.
Here is a list of main acheivements:
A role of new Piwi proteins in Paramecium - my lab discovered that development-specific small RNAs (scnRNAs and iesRNAs) are physically associated with different sets of Piwi proteins in a mutually exclusive manner; the former bind to Ptiwi01 and Ptiwi09, and the latter bind to Ptiwi10 and Ptiwi11.
Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs - In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences.
Dicer-like Enzymes with Sequence Cleavage Preferences - Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.
Epigenetic control of DNA elimination - Another of my studies shows that relative to IES length, there are pronounced differences in the IES sub-terminal base frequencies, suggesting that the IES excisase's recognition/excision preferences change with IES length. My work suggests that some IESs are more difficult to recognize and/or excise than others, thus requiring the greatest targeting support from scnRNAs and iesRNAs. With time, and owing to the effects of purifying selection acting upon IES excisase preferences, longer, epigenetically controlled IESs tend to become shorter and non-epigenetically controlled. There is always demand for scnRNA-dependent IES excision, due to ongoing genomic influxes of invasive DNAs and mutations converting non-epigenetically controlled IESs to epigenetically controlled IESs.
Non-standard genetic codes and genetic code evolution - In addition, I’ve been investigating codon reassignment and evolution of the genetic code using ciliates as models. This diverse group of eukaryotes include multiple examples of non-standard genetic codes which makes them excellent models for this type of investigation. In my recent paper, Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination, 2016 Cell, we describe two codes with efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. The work provided evidence that mRNA 3' ends may contribute to distinguishing stop from sense in a context-dependent manner and further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.
A role of new Piwi proteins in Paramecium - my lab discovered that development-specific small RNAs (scnRNAs and iesRNAs) are physically associated with different sets of Piwi proteins in a mutually exclusive manner; the former bind to Ptiwi01 and Ptiwi09, and the latter bind to Ptiwi10 and Ptiwi11.
Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs - In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences.
Dicer-like Enzymes with Sequence Cleavage Preferences - Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.
Epigenetic control of DNA elimination - Another of my studies shows that relative to IES length, there are pronounced differences in the IES sub-terminal base frequencies, suggesting that the IES excisase's recognition/excision preferences change with IES length. My work suggests that some IESs are more difficult to recognize and/or excise than others, thus requiring the greatest targeting support from scnRNAs and iesRNAs. With time, and owing to the effects of purifying selection acting upon IES excisase preferences, longer, epigenetically controlled IESs tend to become shorter and non-epigenetically controlled. There is always demand for scnRNA-dependent IES excision, due to ongoing genomic influxes of invasive DNAs and mutations converting non-epigenetically controlled IESs to epigenetically controlled IESs.
Non-standard genetic codes and genetic code evolution - In addition, I’ve been investigating codon reassignment and evolution of the genetic code using ciliates as models. This diverse group of eukaryotes include multiple examples of non-standard genetic codes which makes them excellent models for this type of investigation. In my recent paper, Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination, 2016 Cell, we describe two codes with efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. The work provided evidence that mRNA 3' ends may contribute to distinguishing stop from sense in a context-dependent manner and further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.
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.