Periodic Reporting for period 1 - PSEUDOPIN (PSEUDOPIN)
Reporting period: 2022-05-01 to 2024-04-30
RNA contains hundreds of covalent modifications, primarily found in non-coding RNAs such as transfer RNAs (tRNAs) and small nuclear RNAs (snRNAs), though these modifications are also present in messenger RNAs (mRNAs) and small RNAs (sRNAs). These RNA modifications are crucial for genetic regulation, as they influence fundamental RNA properties including stability, structure, and interactions with proteins and other RNAs. This, in turn, regulates essential cellular processes such as splicing and translation.
The project titled "Unraveling the Impact of Pseudouridine RNA Modification upon Plant Immune Activation" (PSEUDOPIN) investigates the role of the RNA modification pseudouridine in plants. Specifically, it explores how changes in the position and abundance of pseudouridine contribute to the genetic reprogramming triggered by pathogen detection. By studying these dynamics, the project aims to elucidate how pseudouridine modification impacts plant immune responses and its broader implications for plant resilience.
Research in this area is of paramount importance due to the current lack of fundamental understanding regarding the role of pseudouridine RNA modification in plant RNAs. There is even less information (nothing) about its function as a key element in regulating gene expression during pathogenicity. The knowledge generated from this project is highly valuable not only to the plant and RNA research communities but also to breeding and seed companies. This understanding could significantly impact the development of resilient crop varieties and advance our comprehension of RNA modifications in plant biology.
The project aims to address two critical questions: (i) uncover the role of RNA pseudouridylation in regulating genetic information in plants, and (ii) analyze how this RNA modification influences plant immunity by impacting key cellular processes such as mRNA splicing and/or translation.
To address these open questions, the objectives of this Marie Skłodowska Curie Action (MSCA) have been:
1) Identify PUS5-dependent Ψ-sites upon immune receptor activation.
2) Investigate the molecular processes, e.g. splicing or translation, affected by PUS5-mediated pseudouridylation.
3) Perform functional analysis of selected PUS5-dependent Ψ sites, if such sites can be identified.
4) Analyse the biological consequences of PUS5-dependent pseudouridylation for the immune response.
The project titled "Unraveling the Impact of Pseudouridine RNA Modification upon Plant Immune Activation" (PSEUDOPIN) investigates the role of the RNA modification pseudouridine in plants. Specifically, it explores how changes in the position and abundance of pseudouridine contribute to the genetic reprogramming triggered by pathogen detection. By studying these dynamics, the project aims to elucidate how pseudouridine modification impacts plant immune responses and its broader implications for plant resilience.
Research in this area is of paramount importance due to the current lack of fundamental understanding regarding the role of pseudouridine RNA modification in plant RNAs. There is even less information (nothing) about its function as a key element in regulating gene expression during pathogenicity. The knowledge generated from this project is highly valuable not only to the plant and RNA research communities but also to breeding and seed companies. This understanding could significantly impact the development of resilient crop varieties and advance our comprehension of RNA modifications in plant biology.
The project aims to address two critical questions: (i) uncover the role of RNA pseudouridylation in regulating genetic information in plants, and (ii) analyze how this RNA modification influences plant immunity by impacting key cellular processes such as mRNA splicing and/or translation.
To address these open questions, the objectives of this Marie Skłodowska Curie Action (MSCA) have been:
1) Identify PUS5-dependent Ψ-sites upon immune receptor activation.
2) Investigate the molecular processes, e.g. splicing or translation, affected by PUS5-mediated pseudouridylation.
3) Perform functional analysis of selected PUS5-dependent Ψ sites, if such sites can be identified.
4) Analyse the biological consequences of PUS5-dependent pseudouridylation for the immune response.
The tasks of this project were subdivided into four distinct work packages (WPs).
WP1 focused on the isolation of specific types of plant RNAs, including ribosomal RNA (rRNA), messenger RNA (mRNA), small RNA (sRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). Using various laboratory techniques, we successfully isolated sRNAs, rRNAs and tRNAs-snRNAs-mRNAs.
WP2 focused on library preparation using a method for pseudouridine detection through Illumina sequencing. We employed the CMC-derivatization method, also known as Pseudo-seq or Ψ-seq, which was originally developed by Carlile and colleagues. This technique enables the mapping of pseudouridine positions with single-nucleotide resolution. The aim of this WP was to generate the raw sequencing data and develop the necessary scripts to map the reads and identify pseudouridine sites. This objective was achieved.
WP3 aimed to elucidate the role of PUS5, the pseudouridine writer, in pseudouridylation. By comparing samples expressing either the short isoform of PUS5, which has a nuclear-cytoplasmic distribution, or the long isoform, which is localized to organelles, we identified sets of genes with pseudouridine sites that vary in abundance depending on which isoform is expressed. This comparison involved detailed analysis of RNA modifications and the impact of different PUS5 isoforms on pseudouridine distribution across the genome. The findings from this WP are still under development, and further research will continue to refine and expand our understanding of PUS5-dependent pseudouridylation. Future work will focus on validating these findings, exploring additional genes and conditions, and integrating this data with other aspects of the project to fully elucidate the role of PUS5 in plant stress responses and gene regulation at the molecular level.
WP4 aimed to investigate the biological consequences of PUS5-dependent pseudouridylation, particularly its impact on plant immunity. This work involved assessing the role of pseudouridine in the immune response by examining how different isoforms of PUS5 (short or long) affect plant resistance to bacterial pathogens, or the growth arrest phenomena. During this WP, we observed that PUS5-dependent pseudouridylation appears to influence plant immunity, with a slight increase in resistance to bacterial pathogens in plants expressing either the short or long isoform of PUS5. The results suggest that while the overall immune response is enhanced, further investigation is needed to determine the specific mechanisms by which pseudouridylation affects pathogen resistance and whether the impact varies between the different PUS5 isoforms. Additionally, although this is preliminary data that requires further verification, the expression of either PUS5 isoform does not appear to influence the growth arrest observed during biotic stress.
During the course of the project, I received extensive training in bioinformatics, particularly in the areas of mapping and identifying RNA modifications, as well as analyzing data related to epitranscriptomics. This training included hands-on experience with various bioinformatics tools and techniques, enhancing my ability to handle and interpret complex sequencing data. Additionally, I received laboratory training focused on library preparation and RNA isolation techniques. This involved mastering protocols for RNA extraction, purification, and the preparation of sequencing libraries, which are crucial for obtaining high-quality data.
Throughout the project, I also supervised two bachelor’s students and two master’s students, guiding them through their research projects and helping them develop their skills. My supervision included mentoring on experimental techniques, data analysis, and research methodologies, contributing to their academic and professional growth. In addition to this, I participated in several group meetings with my supervisor on a weekly basis, as well as monthly institute meetings where we discussed and presented our progress on the project. These meetings were crucial for receiving feedback, brainstorming solutions to challenges, and ensuring the alignment of project goals.
Moreover, I attended RNA-related meetings and conferences to deepen my understanding of the field and stay updated on the latest advancements. These meetings provided valuable opportunities to learn from experts, engage in discussions about recent research findings, and network with other professionals in the field.
Finally, I also collaborated with my supervisor to write two grant applications aimed at securing additional funding and supporting the continuation of our research.
WP1 focused on the isolation of specific types of plant RNAs, including ribosomal RNA (rRNA), messenger RNA (mRNA), small RNA (sRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). Using various laboratory techniques, we successfully isolated sRNAs, rRNAs and tRNAs-snRNAs-mRNAs.
WP2 focused on library preparation using a method for pseudouridine detection through Illumina sequencing. We employed the CMC-derivatization method, also known as Pseudo-seq or Ψ-seq, which was originally developed by Carlile and colleagues. This technique enables the mapping of pseudouridine positions with single-nucleotide resolution. The aim of this WP was to generate the raw sequencing data and develop the necessary scripts to map the reads and identify pseudouridine sites. This objective was achieved.
WP3 aimed to elucidate the role of PUS5, the pseudouridine writer, in pseudouridylation. By comparing samples expressing either the short isoform of PUS5, which has a nuclear-cytoplasmic distribution, or the long isoform, which is localized to organelles, we identified sets of genes with pseudouridine sites that vary in abundance depending on which isoform is expressed. This comparison involved detailed analysis of RNA modifications and the impact of different PUS5 isoforms on pseudouridine distribution across the genome. The findings from this WP are still under development, and further research will continue to refine and expand our understanding of PUS5-dependent pseudouridylation. Future work will focus on validating these findings, exploring additional genes and conditions, and integrating this data with other aspects of the project to fully elucidate the role of PUS5 in plant stress responses and gene regulation at the molecular level.
WP4 aimed to investigate the biological consequences of PUS5-dependent pseudouridylation, particularly its impact on plant immunity. This work involved assessing the role of pseudouridine in the immune response by examining how different isoforms of PUS5 (short or long) affect plant resistance to bacterial pathogens, or the growth arrest phenomena. During this WP, we observed that PUS5-dependent pseudouridylation appears to influence plant immunity, with a slight increase in resistance to bacterial pathogens in plants expressing either the short or long isoform of PUS5. The results suggest that while the overall immune response is enhanced, further investigation is needed to determine the specific mechanisms by which pseudouridylation affects pathogen resistance and whether the impact varies between the different PUS5 isoforms. Additionally, although this is preliminary data that requires further verification, the expression of either PUS5 isoform does not appear to influence the growth arrest observed during biotic stress.
During the course of the project, I received extensive training in bioinformatics, particularly in the areas of mapping and identifying RNA modifications, as well as analyzing data related to epitranscriptomics. This training included hands-on experience with various bioinformatics tools and techniques, enhancing my ability to handle and interpret complex sequencing data. Additionally, I received laboratory training focused on library preparation and RNA isolation techniques. This involved mastering protocols for RNA extraction, purification, and the preparation of sequencing libraries, which are crucial for obtaining high-quality data.
Throughout the project, I also supervised two bachelor’s students and two master’s students, guiding them through their research projects and helping them develop their skills. My supervision included mentoring on experimental techniques, data analysis, and research methodologies, contributing to their academic and professional growth. In addition to this, I participated in several group meetings with my supervisor on a weekly basis, as well as monthly institute meetings where we discussed and presented our progress on the project. These meetings were crucial for receiving feedback, brainstorming solutions to challenges, and ensuring the alignment of project goals.
Moreover, I attended RNA-related meetings and conferences to deepen my understanding of the field and stay updated on the latest advancements. These meetings provided valuable opportunities to learn from experts, engage in discussions about recent research findings, and network with other professionals in the field.
Finally, I also collaborated with my supervisor to write two grant applications aimed at securing additional funding and supporting the continuation of our research.
This MSCA project integrates cutting-edge technologies to explore the role of pseudouridine in plant stress responses. It employs a multidisciplinary approach, combining sequencing technologies with single-base resolution, cell biology, biochemistry, and genetics to uncover the mechanisms by which pseudouridine deposition, mediated by PUS5, influences stress responses.
Innovative aspects include:
1. Development of a workflow for the efficient isolation of specific types of plant RNAs.
2. Use of sequencing technologies for single-base resolution mapping of pseudouridines.
3. Development of scripts and bioinformatics tools for accurate pseudouridine base calling.
4. New methodologies to assess plant growth inhibition and recovery under various stress conditions.
The project's anticipated outcome is to elucidate the role of pseudouridine in plant stress adaptation and determine whether PUS5 is involved in this process. The cutting-edge methodologies employed ensure significant scientific impact and the generation of novel results. Understanding how plants manage stress responses to both biotic and abiotic factors is crucial for seed and breeding companies. This knowledge could facilitate the development of crops that can survive and thrive under various stress conditions highlighting the project’s potential socio-economic value.
Innovative aspects include:
1. Development of a workflow for the efficient isolation of specific types of plant RNAs.
2. Use of sequencing technologies for single-base resolution mapping of pseudouridines.
3. Development of scripts and bioinformatics tools for accurate pseudouridine base calling.
4. New methodologies to assess plant growth inhibition and recovery under various stress conditions.
The project's anticipated outcome is to elucidate the role of pseudouridine in plant stress adaptation and determine whether PUS5 is involved in this process. The cutting-edge methodologies employed ensure significant scientific impact and the generation of novel results. Understanding how plants manage stress responses to both biotic and abiotic factors is crucial for seed and breeding companies. This knowledge could facilitate the development of crops that can survive and thrive under various stress conditions highlighting the project’s potential socio-economic value.