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.
