A multidimensional study of dsRNA binding proteins DHX9 and ADAR1

• Applicant: Tugce Aktas, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 456668871

Majority of the eukaryotic genomes is made up of non-coding DNA which provide complex regulatory mechanisms to gene expression. Transposable elements (TEs) make up half of the human genome and get expressed as the coding DNA is being transcribed. Therefore, gene regulatory mechanisms cannot be decoupled from the presence of highly abundant and repeated TEs. There is substantial evidence for the role of TEs in chromatin regulation, however their roles in co-transcriptional and post-transcriptional RNA processing remains largely unknown. In the course of evolution, TE insertions happen in waves where their copy numbers suddenly increase in the host genome. During these invasion events, new RNA-binding proteins (RBPs) can evolve or existing ones can be co-opted to suppress potential disruptive effects of harboring many transposable element insertions within protein-coding genes. We identified the RNA-helicase; DHX9 and the RNA-editase ADAR1 as two double stranded RNA (dsRNA) binding proteins exerting their enzymatic activity on repeat derived and highly structured RNAs. These proteins both work on the same substrate in humans (Alu-Alu pairs) and they unwind secondary structures in RNAs through their enzymatic activities. Furthermore, DHX9 interacts with mouse SINE-B repeats which evolved divergently from primate Alu repeats, showing that the primary target of DHX9 is the most abundant dsRNA species in an organism. In the absence of DHX9, we scored RNA processing defects ranging from missplicing such as back-splicing leading to increased circular RNA production, to transcription termination defects and translational suppression of mRNA with inverted Alu elements at their 3’UTRs. In our biochemical interactome analysis, we identified the interferon induced isoform of ADAR1 (p150) as a direct interaction partner of DHX9 and showed that this interaction is conserved from rodents to primates, indicating that it has evolved preceding the divergence of SINE repeats, potentially against viral invaders. In this proposal, we suggest an integrated and multidimensional analysis of DHX9 and ADAR1 and their role in allowing the expansion of TEs across evolution. We will start with the analysis of temporally resolved RNA processing defects to be able to understand the order of events and their dependence on DHX9 and/or ADAR. We will further perform unbiased screens to identify the network of RBPs working in cooperation with DHX9 and ADAR1 to assure proper RNA processing in the nucleus by using multiple genomics methods. Ultimately, we will generate RNA topology data upon depletion of DHX9 and/or ADAR1 and build an integrative model with all other data generated in this project. This study will generate one of the most comprehensive datasets that characterize the complex network of dsRNA and RBPs in the nucleus and will shed light on the impact of RBPs that target tranposon-derived RNA on genome evolution.

• DFG Programme: Research Grants