Periodic Reporting for period 4 - PlasmoSilencing (Exoribonuclease-mediated degradation of nascent RNA in Malaria Parasites: A Novel Mechanism in Virulence Gene Silencing)
Berichtszeitraum: 2020-05-01 bis 2020-10-31
Our knowledge on malaria parasite posttranscriptional factors controlling expression of genes linked to severe malaria remains very limited. Our ground-breaking discovery of a mechanism that is a central player in this process via a new type of RNase-mediated gene silencing mechanism, has opened several new avenues linking news aspects of RNA biology to malaria virulence. This emerging topic represents the main focus of PlasmoSilencing. Malaria remains a major burden in many endemic countries and new insight into the underlying molecular events of the pathogenesis and transmission process may lead to intervention strategies that could avoid the development of severe malaria and spreading of the disease. The ambitious objectives of this project aim to establish new conceptual findings in the field of control of gene expression by a exploring an emerging field called epitranscriptomics. Furthermore, this mostly unexplored field has the potential of translational aspects for the development of new intervention strategies against malaria.
In this project, we have addressed the major objectives defined in this project: i) the molecular mechanism of exoribonuclease-mediated RNA degradation in plasmodial gene expression, ii) the role of PfRNase II transcript levels in virulence gene expression that are linked to severe malaria, iii) the role of exoribonucleases Dis3 and Rrp6 in posttranscriptional gene regulation.
Taken together, our work has resulted in 17 publications with support of this grant and two manuscripts are in preparation that will be published in 2021. This includes work directly linked to the main objectives of PlasmoSilencing and work aimed to develop new methods to overcome technical hurdles in chromatin analysis and genome editing of P. falciparum. Due to space limits, only some of the achievements will be mentioned here. One highlight is that the exoribonuclease-controlled ncRNA gene family (called GC-rich element) acts in trans as an enhancer-like ncRNA in monoallelic expression of virulence genes. A second discovery shows, that a second type of 3’-5’ exoribonuclease (Dis3) controls mainly antisense RNA, that is an emerging regulator of gene expression in malaria parasites. A third highlight is the identification of mRNA adenosine methylation as a regulator of mRNA stability and translation in P. falciparum. One of the objectives, the role of RNase II transcript levels in virulence gene regulation, could not be completed, due to technical problems to establish a genetic system to dose the transcript levels of a given gene in P. falciparum.
Taken together, our work has resulted in 17 publications with support of this grant and two manuscripts are in preparation that will be published in 2021. This includes work directly linked to the main objectives of PlasmoSilencing and work aimed to develop new methods to overcome technical hurdles in chromatin analysis and genome editing of P. falciparum. Due to space limits, only some of the achievements will be mentioned here. One highlight is that the exoribonuclease-controlled ncRNA gene family (called GC-rich element) acts in trans as an enhancer-like ncRNA in monoallelic expression of virulence genes. A second discovery shows, that a second type of 3’-5’ exoribonuclease (Dis3) controls mainly antisense RNA, that is an emerging regulator of gene expression in malaria parasites. A third highlight is the identification of mRNA adenosine methylation as a regulator of mRNA stability and translation in P. falciparum. One of the objectives, the role of RNase II transcript levels in virulence gene regulation, could not be completed, due to technical problems to establish a genetic system to dose the transcript levels of a given gene in P. falciparum.
In order to overcome existing hurdles, we had to develop a number of new methods for malaria parasites. A main issue is to silence an entire ncRNA multigene family. Here we used the dead Cas9 to target regulatory sequences common to all gene family members (15). After a number of attempts we developed protocols that now work to target specific genome regions either for pull down of chromatin associated to specific regions of interest or to inactivate gene transcription by interfering with the Pol II and Pol III transcription. This methodology has revealed a function for the PfRNase controlled ncRNA gene family as enhancer of monoallelic expression of the major virulence gene family (var gene). Another breakthrough is the discovery of unusual high adenosine methylation in mRNA (m6A) of P. falciparum, the identification of reader proteins for this epigenetic mark and the role of this mark in developmental progression of the blood stage cycle. This latter observation opens a new field in epitranscriptomics for this pathogen with a focus on translational repression and pathogen transmission via the mosquito vector.
Final conclusions: The initial project outlined for PlasmoSilencing has not been always had a linear evolution due to different technical challenges. At least two of the three aims have been moved forward and resulted in fundamental new insight into the exoribonuclease regulators of RNA (ncRNA and mRNA) degradation and RNA modifications (mainly m6A) that modulate the RNA. Technical challenges were often the motivation to overcome roadblocks for this pathogen, that is far from being a model system and difficult to manipulate. We developed dCas9 genome engineering tools to isolate locus specific chromatin components and to shut down multiple promoters from a gene family. Furthermore, we adapted for the first time a method, to identify RNA-binding proteins from native RNA-protein complexes using a method called ChIRP (chromatin isolation by RNA isolation). These methods put us in a unique situation to further explore the precise molecular events that involve ncRNA activity in virulence gene expression. In conclusion, PlasmoSilencing has had a huge impact in the field of exoribonuclease/ncRNA/mRNA biology of P. falciparum. It has helped to move this field to an unprecedented level of insight.
As a result of the high scientific output, one postdoc (S. Baumgarten) has further developed one emerging aspect of PlasmoSilencing and has obtained in 2020 an ERC Starting grant to explore as group leader at the Institut Pasteur the role of RNA modifications in P. falciparum. Another postdoc who has been hired to work on PlasmoSilencing, has obtained a staff position (Charge de Recherche) at the Institut Pasteur to build a new team on plasmodial chromatin biology (J. Bryant). In addition, two students financed by this project have obtained their PhD in 2019 and continue to work in the field of molecular parasitology (A. Barcons Simmons & E. Hammam). Last but not least, new grants have been obtained in 2020 on results obtained from PlasmoSilencing.
Final conclusions: The initial project outlined for PlasmoSilencing has not been always had a linear evolution due to different technical challenges. At least two of the three aims have been moved forward and resulted in fundamental new insight into the exoribonuclease regulators of RNA (ncRNA and mRNA) degradation and RNA modifications (mainly m6A) that modulate the RNA. Technical challenges were often the motivation to overcome roadblocks for this pathogen, that is far from being a model system and difficult to manipulate. We developed dCas9 genome engineering tools to isolate locus specific chromatin components and to shut down multiple promoters from a gene family. Furthermore, we adapted for the first time a method, to identify RNA-binding proteins from native RNA-protein complexes using a method called ChIRP (chromatin isolation by RNA isolation). These methods put us in a unique situation to further explore the precise molecular events that involve ncRNA activity in virulence gene expression. In conclusion, PlasmoSilencing has had a huge impact in the field of exoribonuclease/ncRNA/mRNA biology of P. falciparum. It has helped to move this field to an unprecedented level of insight.
As a result of the high scientific output, one postdoc (S. Baumgarten) has further developed one emerging aspect of PlasmoSilencing and has obtained in 2020 an ERC Starting grant to explore as group leader at the Institut Pasteur the role of RNA modifications in P. falciparum. Another postdoc who has been hired to work on PlasmoSilencing, has obtained a staff position (Charge de Recherche) at the Institut Pasteur to build a new team on plasmodial chromatin biology (J. Bryant). In addition, two students financed by this project have obtained their PhD in 2019 and continue to work in the field of molecular parasitology (A. Barcons Simmons & E. Hammam). Last but not least, new grants have been obtained in 2020 on results obtained from PlasmoSilencing.