Periodic Reporting for period 1 - EXOonRNA (Delineating the kinetic RNA interactome of nuclear exosome adaptor complexes)
Berichtszeitraum: 2022-09-01 bis 2024-08-31
The human genome is transcribed extensively, but most transcription events do not result in functional RNA molecules. Therefore, much of the RNA output of the genome is regulated at the level of RNA degradation, a key gene regulatory mechanism that remains significantly understudied. Cells must distinguish functional RNAs from non-functional ones and efficiently eliminate excess transcripts, which would otherwise interfere with other RNA-related processes. The RNA exosome complex is the primary molecular machine responsible for degrading newly produced RNA in eukaryotic nuclei (Figure 1a). It filters out RNAs marked for degradation from their functional counterparts with the help of adapter complexes, specifically the nuclear exosome targeting (NEXT) complex and the polyA-tail exosome targeting (PAXT) connection. Both of these adapter complexes consist of several RNA-binding proteins (RBPs) that interact with RNA, but exactly how they distinguish functional from non-functional RNA remains a mystery in molecular biology. This makes it crucial to uncover the principles by which nuclear exosome adaptors target RNA. This need is emphasized by findings that abnormal RNA levels are strongly linked to human diseases and that both nuclear RNA degradation and ExoAC complexes are connected to disease biology. Therefore, sorting nuclear RNA for degradation is a fundamental challenge in gene regulation with important implications for biomedicine. Moreover, with the rise of RNA-based technologies, such as mRNA vaccines, a deep understanding of RNA decay pathways is essential for future advancements in these areas.
In the proposed project, I aimed to identify critical checkpoints in nuclear RNA biogenesis that help distinguish functional from non-functional RNA. To achieve this, I have studied the RNA-binding components of the NEXT and PAXT ExoACs, namely RBM7 and LENG8 proteins, using a crosslinking followed by immunoprecipitation (CLIP) method (Objective 1). I hypothesize that to promote RNA decay via the nuclear exosome, the RNA-binding components of ExoACs assemble on RNA substrates in a stepwise manner, ultimately marking the substrate for degradation. To test this, I analyzed RNA-binding maps of key proteins associated with the nuclear exosome adapter complexes (Objective 2). On the basis of above research work I concluded the key principles how two different ExoAC complexes NEXT and PAXT target RNAs in the nucleus (Figure 1b). Namely, I found that NEXT interacts with short non-polyadenylated RNAs co-transcriptionally, just after their synthesis. Conversely, PAXT approaches polyadenylated RNAs at the late stages of their biogenesis in the nucleus, where it competes with export machinery for RNAs as parts of ribonucleoprotein particles (RNPs) with shared features with export-competent RNPs. This results shed the light on the features of RNA but also interacting RBPs, associated either with nuclear decay or export to the cytoplasm.
In the proposed project, I aimed to identify critical checkpoints in nuclear RNA biogenesis that help distinguish functional from non-functional RNA. To achieve this, I have studied the RNA-binding components of the NEXT and PAXT ExoACs, namely RBM7 and LENG8 proteins, using a crosslinking followed by immunoprecipitation (CLIP) method (Objective 1). I hypothesize that to promote RNA decay via the nuclear exosome, the RNA-binding components of ExoACs assemble on RNA substrates in a stepwise manner, ultimately marking the substrate for degradation. To test this, I analyzed RNA-binding maps of key proteins associated with the nuclear exosome adapter complexes (Objective 2). On the basis of above research work I concluded the key principles how two different ExoAC complexes NEXT and PAXT target RNAs in the nucleus (Figure 1b). Namely, I found that NEXT interacts with short non-polyadenylated RNAs co-transcriptionally, just after their synthesis. Conversely, PAXT approaches polyadenylated RNAs at the late stages of their biogenesis in the nucleus, where it competes with export machinery for RNAs as parts of ribonucleoprotein particles (RNPs) with shared features with export-competent RNPs. This results shed the light on the features of RNA but also interacting RBPs, associated either with nuclear decay or export to the cytoplasm.
To address the overall aim and specific objectives of the project, I followed the proposed implementation plan where all activities and tasks were divided into the logical work packages.
To perform RNA-protein interaction profiling of components of exosome adapter complexes (WP1), I established an experimental system using CRISPR-mediated endogenous tagging in human cell lines. This included generating new human cell lines expressing tagged components of exosome adapter complexes. I then optimized and conducted CLIP experiments on nuclear RNA turnover factors, performed RNA sequencing under conditions of factor depletion, and gathered additional experimental data.
Next, to analyze and interpret the data obtained(WP2), I performed computational analyses of CLIP and RNA sequencing datasets. I tested the research hypothesis using complementary experimental approaches, including co-immunoprecipitation of the relevant proteins, rapid depletion of exosome adapter complex components in human cells, and subsequent transcriptome sequencing followed by differential expression analysis.
During the research activities, I underwent training relevant to the project's objectives, including hands-on laboratory training in the T-CLIP method and bioinformatics training for data analysis (WP3). Additionally, I participated in career development training provided by the host institution, including seminars on academic writing and presentations. I also improved my supervision skills by assisting a PhD student with a related early-stage technical project, and developed project management skills by executing the project and attending an EMBO Research Project Management course.
Throughout the project, I managed its overall progress, including preparing for project status meetings and overseeing financial reporting. I also engaged in dissemination activities by presenting the project at institutional, national, and international meetings, including an oral presentation at the 2024 Annual RNA Society Meeting in Edinburgh. Furthermore, I prepared manuscripts for publication, a process that is ongoing (WP4).
As a key outcome of the project, I identified a novel component of the PAXT connection—the LENG8-PCID2 module, which is involved in the nuclear retention and degradation of processed and polyadenylated RNAs, similar to functional mRNAs. LENG8 is an RNA-binding component of PAXT that acts through its interaction with packaged RNPs. Together with a group of collaborators, we further discovered that, due to the RNA-binding activity of LENG8 and its biochemical similarity to the nuclear export component GANP, PAXT competes with nuclear export for RNA substrates. These results have significant implications not only for the field of nuclear RNA decay but also for the study of nuclear export. Another important result of this work is the acquisition of RNA-binding profiles of the RNA-binding components of the major nuclear exosome adaptor complexes, NEXT and PAXT. These profiles have been revealed and can now be further analyzed and compared with other key RNA turnover regulators, such as nuclear export factors. The ExoAC RNA-binding profiles uncovered distinct mechanisms by which the nuclear degradation machinery targets short non-polyadenylated transcripts and longer processed RNAs (Figure 1b). The results of this work were presented at several national and international scientific meetings, including the RNA Society and the Danish RNA Society meetings. The findings will be disseminated through scientific papers, which are currently being prepared for publication.
To perform RNA-protein interaction profiling of components of exosome adapter complexes (WP1), I established an experimental system using CRISPR-mediated endogenous tagging in human cell lines. This included generating new human cell lines expressing tagged components of exosome adapter complexes. I then optimized and conducted CLIP experiments on nuclear RNA turnover factors, performed RNA sequencing under conditions of factor depletion, and gathered additional experimental data.
Next, to analyze and interpret the data obtained(WP2), I performed computational analyses of CLIP and RNA sequencing datasets. I tested the research hypothesis using complementary experimental approaches, including co-immunoprecipitation of the relevant proteins, rapid depletion of exosome adapter complex components in human cells, and subsequent transcriptome sequencing followed by differential expression analysis.
During the research activities, I underwent training relevant to the project's objectives, including hands-on laboratory training in the T-CLIP method and bioinformatics training for data analysis (WP3). Additionally, I participated in career development training provided by the host institution, including seminars on academic writing and presentations. I also improved my supervision skills by assisting a PhD student with a related early-stage technical project, and developed project management skills by executing the project and attending an EMBO Research Project Management course.
Throughout the project, I managed its overall progress, including preparing for project status meetings and overseeing financial reporting. I also engaged in dissemination activities by presenting the project at institutional, national, and international meetings, including an oral presentation at the 2024 Annual RNA Society Meeting in Edinburgh. Furthermore, I prepared manuscripts for publication, a process that is ongoing (WP4).
As a key outcome of the project, I identified a novel component of the PAXT connection—the LENG8-PCID2 module, which is involved in the nuclear retention and degradation of processed and polyadenylated RNAs, similar to functional mRNAs. LENG8 is an RNA-binding component of PAXT that acts through its interaction with packaged RNPs. Together with a group of collaborators, we further discovered that, due to the RNA-binding activity of LENG8 and its biochemical similarity to the nuclear export component GANP, PAXT competes with nuclear export for RNA substrates. These results have significant implications not only for the field of nuclear RNA decay but also for the study of nuclear export. Another important result of this work is the acquisition of RNA-binding profiles of the RNA-binding components of the major nuclear exosome adaptor complexes, NEXT and PAXT. These profiles have been revealed and can now be further analyzed and compared with other key RNA turnover regulators, such as nuclear export factors. The ExoAC RNA-binding profiles uncovered distinct mechanisms by which the nuclear degradation machinery targets short non-polyadenylated transcripts and longer processed RNAs (Figure 1b). The results of this work were presented at several national and international scientific meetings, including the RNA Society and the Danish RNA Society meetings. The findings will be disseminated through scientific papers, which are currently being prepared for publication.
The assembly of exosome adapter complexes on newly produced RNA is of significant interest to the broader RNA research community. The data collected in this project could be further analyzed alongside earlier and future CLIP datasets, generating additional research insights and impacting the field for years to come.
Moreover, the project spans several areas of biological science. RNA turnover control is crucial for maintaining normal cell function and overall organismal health. Mutations in nuclear exosome core subunits and ExoAC components have been linked to neurological disorders and cancer. Therefore, this study will provide valuable insights into RNA turnover mechanisms that could help unravel the underlying causes of these diseases in the future.
Moreover, the project spans several areas of biological science. RNA turnover control is crucial for maintaining normal cell function and overall organismal health. Mutations in nuclear exosome core subunits and ExoAC components have been linked to neurological disorders and cancer. Therefore, this study will provide valuable insights into RNA turnover mechanisms that could help unravel the underlying causes of these diseases in the future.