Periodic Reporting for period 4 - RiboTrace (Bridging temporal resolution gaps to dissect RNA silencing at the molecular and genomic scale)
Reporting period: 2023-08-01 to 2025-01-31
Gene regulation is the fundamental process that controls genome function and it pervades most biology, from organismal development to cellular differentiation and physiological responses to external stimuli and pathogens. To this end, gene expression is controlled at multiple levels, including RNA transcription, processing, translation and stability and deviations in most of these aspects invoke detrimental consequences to organisms and represent a major cause for human disease.
We aim to study the mechanisms of RNA silencing, the least understood aspect of gene regulation. We bridge temporal resolution gaps to unravel the fundamental biological principles of RNA decay at the molecular and genomic scale. Our goal is to understand how the quality and quantity of the transcriptome is controlled at the molecular level in flies and mammals. Our studies will [1] provide insights into the emerging role of RNA modifications in the regulation of RNA fate and function, [2] determine possible causes for aberrant gene expression profiles that have been associated with human diseases, such as the Perlman Syndrome; and [3] establish technologies that unravel the molecular signatures of RNA decay, as well as the underlying gene silencing modules and their organization in pathways at the genomic scale.
In this proposal, we combine diverse scientific approaches and experimental techniques to dissect the regulation of gene expression at the molecular and genomic scale in flies and mammals. To this end, we unravel the molecular basis, physiological targets and biological functions of uridylation-triggered RNA silencing; we identify and dissect fundamental principles in global and transcript-specific RNA decay; and we establish novel tools to unravel the molecular signatures, and mechanistic components of RNA turnover at the genomic scale. Throughout, we link our results back to the established function of RNA silencing in the regulation of organismal development, physiology and disease. In summary, our work will significantly advance our understanding of post-transcriptional gene regulation, a phenomenon with enormous biological and biomedical relevance.
We aim to study the mechanisms of RNA silencing, the least understood aspect of gene regulation. We bridge temporal resolution gaps to unravel the fundamental biological principles of RNA decay at the molecular and genomic scale. Our goal is to understand how the quality and quantity of the transcriptome is controlled at the molecular level in flies and mammals. Our studies will [1] provide insights into the emerging role of RNA modifications in the regulation of RNA fate and function, [2] determine possible causes for aberrant gene expression profiles that have been associated with human diseases, such as the Perlman Syndrome; and [3] establish technologies that unravel the molecular signatures of RNA decay, as well as the underlying gene silencing modules and their organization in pathways at the genomic scale.
In this proposal, we combine diverse scientific approaches and experimental techniques to dissect the regulation of gene expression at the molecular and genomic scale in flies and mammals. To this end, we unravel the molecular basis, physiological targets and biological functions of uridylation-triggered RNA silencing; we identify and dissect fundamental principles in global and transcript-specific RNA decay; and we establish novel tools to unravel the molecular signatures, and mechanistic components of RNA turnover at the genomic scale. Throughout, we link our results back to the established function of RNA silencing in the regulation of organismal development, physiology and disease. In summary, our work will significantly advance our understanding of post-transcriptional gene regulation, a phenomenon with enormous biological and biomedical relevance.
The ERC consolidator grant “RiboTrace” is a major external funding source of the Ameres lab and aims to dissect the regulation of gene expression at the molecular and genomic scale in flies and mammals. The ERC Consolidator proposal formulated the following two major aims:
1) To dissect the mechanisms and biological function of uridylation-triggered gene silencing
2) To elucidate the fundamental principle of RNA silencing at the genomic scale
The proposed projects are well on their way and initial results have been published (see below). Progress on the specific aims were made as follows:
Aim 1: We have established and employed a high-throughput sequencing-compatible biochemical assay to interrogate TRUMP-reaction kinetics, resulting in a comprehensive dataset on the molecular principles underlying uridylation-triggered RNA decay in flies (Aim1.1). A cell-lysate-based approach to determine endogenous target RNAs of the TRUMP complex has been established and is currently applied to different cell-type- and tissue-lysates (Aim 1.2). The mRNA tethering system to address Tailor’s function in mRNA regulation has been set up and revealed initial insights into the repression of gene expression by translational repression and mRNA turnover. Furthermore, a genetic dependency screen and biochemical interaction studies of TUTases have been executed in the mammalian system, revealing candidate factors that are currently being evaluated (Aim 1.3). A comprehensive study on the function of the TRUMP complex in promoting antiviral silencing resulted in the identification of an enigmatic multiple-turnover factor for RNA interference in flies and the manuscript is currently being finalized for publication (Aim 1.4). Beyond those defined aims, we were also able to establish structure-function relationship for microRNA 3´ end trimming by Nibbler (Xie et al., 2020).
Aim 2: We have established enhanced protocols for the transcriptome-wide characterization of gene expression kinetics, including transcriptional output and RNA turnover (Aim 2.1). These protocols contributed significantly to resolving the hijacking of transcriptional condensates by endogenous retroviruses (Asimi et al., 2022) and the role of NMD in timely cell fate transitions (Huth et al., 2022). Furthermore, we developed time-resolved mRNA sequencing strategies in vivo in zebrafish embryos, which resoved the kinetics of maternal and zygotic gene expression in early zebrafish development (Bhat et al., 2022). The genetic tools and protocols for the systematic dissection of (m)RNA decay activities have been put in place and inducible CRISPR knockout strategies have enabled to provide a first definitive view on the cellular essentiality of RNA decay enzymes and their assembly into multi-component complexes in mammals, such as the RNA exosome (Aim 2.2). Furthermore, we have established ORF-trap, a novel genomics approach to interrogate the mRNA-regulatory proteome in mammals, revealing novel putative gene regulatory proteins which we are currently following up on (Aim 2.3).
1) To dissect the mechanisms and biological function of uridylation-triggered gene silencing
2) To elucidate the fundamental principle of RNA silencing at the genomic scale
The proposed projects are well on their way and initial results have been published (see below). Progress on the specific aims were made as follows:
Aim 1: We have established and employed a high-throughput sequencing-compatible biochemical assay to interrogate TRUMP-reaction kinetics, resulting in a comprehensive dataset on the molecular principles underlying uridylation-triggered RNA decay in flies (Aim1.1). A cell-lysate-based approach to determine endogenous target RNAs of the TRUMP complex has been established and is currently applied to different cell-type- and tissue-lysates (Aim 1.2). The mRNA tethering system to address Tailor’s function in mRNA regulation has been set up and revealed initial insights into the repression of gene expression by translational repression and mRNA turnover. Furthermore, a genetic dependency screen and biochemical interaction studies of TUTases have been executed in the mammalian system, revealing candidate factors that are currently being evaluated (Aim 1.3). A comprehensive study on the function of the TRUMP complex in promoting antiviral silencing resulted in the identification of an enigmatic multiple-turnover factor for RNA interference in flies and the manuscript is currently being finalized for publication (Aim 1.4). Beyond those defined aims, we were also able to establish structure-function relationship for microRNA 3´ end trimming by Nibbler (Xie et al., 2020).
Aim 2: We have established enhanced protocols for the transcriptome-wide characterization of gene expression kinetics, including transcriptional output and RNA turnover (Aim 2.1). These protocols contributed significantly to resolving the hijacking of transcriptional condensates by endogenous retroviruses (Asimi et al., 2022) and the role of NMD in timely cell fate transitions (Huth et al., 2022). Furthermore, we developed time-resolved mRNA sequencing strategies in vivo in zebrafish embryos, which resoved the kinetics of maternal and zygotic gene expression in early zebrafish development (Bhat et al., 2022). The genetic tools and protocols for the systematic dissection of (m)RNA decay activities have been put in place and inducible CRISPR knockout strategies have enabled to provide a first definitive view on the cellular essentiality of RNA decay enzymes and their assembly into multi-component complexes in mammals, such as the RNA exosome (Aim 2.2). Furthermore, we have established ORF-trap, a novel genomics approach to interrogate the mRNA-regulatory proteome in mammals, revealing novel putative gene regulatory proteins which we are currently following up on (Aim 2.3).
The post-transcriptional addition of nucleotides to the 3 ́ end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. While increasing evidence supports a broad and conserved role of 3 ́ terminal nucleotide additions in the regulation of RNA fate and function, the molecular mechanisms, cellular targets and biological functions of RNA uridylation remain largely unexplored. We will employ high- throughput biochemical reconstitution assays in order to establish the molecular basis for uridylation-directed RNA decay; and we will combine genome engineering and targeted RNA sequencing approaches, as well as functional genomics in fly and mammalian cells to unravel the molecular substrates and biological functions of RNA uridylation-mediated gene silencing.
Understanding the molecular principles of RNA turnover at the genomic scale has so far been obstructed by the limited resolution of available approaches. What are the molecular features, components and time-scales of mammalian RNA silencing pathways? And how does mammalian RNA metabolism link to RNA 3 ́ uridylation to establish gene silencing? We will establish time-resolved RNA sequencing across entire transcriptomes to obtain a global view on mammalian RNA turnover and its molecular signatures; and we will combine this approach with chemical-genetic protein depletion approaches to deconvolute the primary activity of major RNA metabolic enzymes in mammals. Finally, we will apply haploid genetics in mESCs to uncover genetic interactors of TUTase-directed mRNA silencing in mammals and we will apply functional genomics to systematically interrogate the mammalian proteome for its mRNA silencing potential.
Understanding the molecular principles of RNA turnover at the genomic scale has so far been obstructed by the limited resolution of available approaches. What are the molecular features, components and time-scales of mammalian RNA silencing pathways? And how does mammalian RNA metabolism link to RNA 3 ́ uridylation to establish gene silencing? We will establish time-resolved RNA sequencing across entire transcriptomes to obtain a global view on mammalian RNA turnover and its molecular signatures; and we will combine this approach with chemical-genetic protein depletion approaches to deconvolute the primary activity of major RNA metabolic enzymes in mammals. Finally, we will apply haploid genetics in mESCs to uncover genetic interactors of TUTase-directed mRNA silencing in mammals and we will apply functional genomics to systematically interrogate the mammalian proteome for its mRNA silencing potential.