Poster Session 5: RNA Modification & Condensation 21:00 - 22:00 Wednesday, 27th May, 2020 Poster 53 Chemo-enzymatic synthesis of site-specifically modified and photo-caged RNAs Bozana Knezic1,2, Sara Keyhani1,2, Alexey Sudakov1,2, Alexander Heckel1, Harald Schwalbe1,2 1Goethe University Frankfurt, Frankfurt, Germany. 2Center for Biomolecular Magnetic Resonance (BMRZ), Frankfurt, Germany Abstract Posttranscriptional editing of RNAs occurs in all types of cells, often for regulatory purposes. Editing includes splicing and ligation reactions affecting larger parts of the RNA. Other types of modifications are the deamination or methylation of single nucleobases affecting smaller RNA regions. Such reactions happen, for instance, with A (to I) and C (to U). These modifications impact the sequence and may alter the secondary structure of the RNA. Furthermore, they can lead to a different amino acid sequence and can ultimately affect the translation efficiency and diversify the cellular phenotype. In addition, there is evidence that posttranscriptional modifications affect liquid-liquid phase separation (LLPS) and as a consequence could participate in the support or even prevention of various diseases. In order to conduct experiments designed to investigate the impact of posttranscriptional editing, preparation of site-specifically modified RNAs is absolutely essential. For this purpose, solid-phase synthesis is the favored method up to this day. However, the technique is limited in terms of length of the desired RNA construct to a maximum of approximately 50 nt, if pure samples in sufficient quantities are required. To overcome this limitation, this project considers a chemo-enzymatic RNA synthesis approach[1]. This method was developed in our labs and allows the site-specific incorporation of modified nucleotides into a target RNA of virtually unlimited length. We were able to incorporate particular modified nucleotides into different long RNAs. This includes inosine and other naturally occurring nucleotides, fluorinated nucleotides and nucleotides tagged with photolabile-protecting groups. The intention of this study is to develop a library of modified nucleotides, which are compatible with the before-mentioned synthesis approach. This approach could significantly support other methods and projects involved in RNA sequencing and modification recognition. In this case, a given site- specifically modified RNA could serve as a bench-mark for newly designed sequencing methods. [1] S. Keyhani, T. Goldau, A. Blümler, A. Heckel, H. Schwalbe, Angew. Chemie Int. Ed. 2018, 57, 12017–12021. Presenting author email [email protected] Topic category RNA Modification & Editing 76 m6A restricts axonal growth in Drosophila through modulation of Fragile X mental retardation protein target selection Alessia Soldano1, Lina Worpenberg2, Chiara Paolantoni2, Sara Longhi1, Miriam Mulorz3, Tina Lence3, Hans- Hermann Wessels4, Giuseppe Aiello1, Michela Notarangelo1, FX Reymond Sutandy3, Marion Scheibe3, Rhagu R. Edupuganti5, Anke Busch6, Martin M. Möckel7, Michiel Vermeulen5, Falk Butter3, Uwe Ohler4, Christoph Dieterich8, Alessandro Quattrone1, Jean-Yves Roignant 2 1Centre for Integrative Biology, University of Trento, Trento, Italy. 2Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. 3Institute of Molecular Biology, Mainz, Germany. 4Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany. 5Radboud Institute for Molecular Life Sciences, Oncode Institute, Nijmegen, Netherlands. 6Bioinformatics Core Facility, IMB, Mainz, Germany. 7Protein Production Core Facility, IMB, Mainz, Germany. 8Klaus Tschira Institute for Integrative Computational Cardiology and Department of Internal Medicine III, University Hospital Heidelberg; German Center for Cardiovascular Research (DZHK), Heidelberg, Germany Abstract The abundant mRNA modification N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. m6A is particularly enriched in the nervous system of several species and its dysregulation has been associated with neurodevelopmental defects as well as neural dysfunctions. In Drosophila, the loss of m6A alters fly behavior but the underlying mechanism and the role of m6A during nervous system development has remained elusive. Here we found that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions, as well as, in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system required for limiting axonal growth. Mechanistically, we show that Ythdf interacts directly with Fragile X mental retardation protein (Fmr1) to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection. Presenting author email [email protected] Topic category RNA Modification & Editing 209 Programmable RNA base editing with a single engineered protein Wenjiang Han1,2, Wendi Huang1,2, Tong Wei1,2, Yanwen Ye1,2, Miaowei Mao1, Zefeng Wang1 1CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Sciences, Shanghai, China. 22.University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China Abstract Programmable base editing of RNA enables re-writing genetic code on specific sites. Current tools for specific RNA editing dependent on the assembly or recruitment of the guide RNA into an RNA/protein complex, which may cause delivery barrier, low editing efficiency, and high immunogenicity. Here, we report a new set of tools, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. The REWIRE system contains a human-originated programmable RNA-binding domain (PUF domain) to specifically recognize its target and ADAR or Apobec3A deaminase domain to achieve A-to-I or C-to- U editing. Using this system, we achieved up to 80% editing efficiency in A-to-I editing and 60% efficiency in C- to-U editing, with little non-specific editing in the targeted region and a low level of off-target effect globally. We were able to apply the REWIRE system to rescue the disease-associated base mutations and to modify mitochondrial RNAs. The RNA binding module of this system was further optimized to increase the editing specificity and minimize off-target effects. As a single-component base editing system originated form human proteins, REWIRE presents a precise and efficient RNA-editing platform with potential in basic research and gene therapy. Presenting author email [email protected] Topic category RNA Modification & Editing 226 C/D Box RNAs protect tRNA Met(e) from stress-induced clivage Patrice Vitali, Tamàs Kiss CBI-CNRS; Université Paul Sabatier, Toulouse, France Abstract Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble cytidine of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments. Presenting author email [email protected] Topic category RNA Modification & Editing 274 Transcriptome-wide analysis of ADAR3 binding and the role of ADAR3 in increasing MAVS activity in response to double-stranded RNA Reshma Raghava Kurup, Emilie Oakes, Aidan Manning, Pranathi Vadlamani, Heather Hundley Indiana University, Bloomington, IN, USA Abstract RNA editing contributes to transcriptome diversity by influencing regulatory processes such as RNA splicing, stability, localization and translational efficiency. Adenosine-to-inosine (A-to-I) editing is one of the most abundant types of RNA editing in humans and is carried out by ADARs. In humans, there are three ADAR family members, ADAR1, ADAR2 and ADAR3. ADAR3 is less studied due to brain-specific expression and lack of known editing activity. Recently our lab demonstrated that ADAR3 is elevated in glioblastomas compared to adjacent normal tissue and binding of ADAR3 to one neuronal transcript, GRIA2, leads to inhibition of editing by ADAR2. However, the role of ADAR3 in the regulation of editing and gene expression in glioblastoma is not well understood. A transcriptome-wide analysis was performed to determine RNA targets of ADAR3 and the effects of ADAR3 on RNA editing in the U87 glioblastoma cell line. Using RIP-seq analysis, 3316 ADAR3 bound targets were identified. I found that ADAR3 expression resulted in differential editing and gene expression transcriptome- wide. Around 80% of sites have reduced editing and the majority of differential edited sites are located in 3’UTRs. In this study, I showed that ADAR3 act as a negative regulator of ADAR1-mediated editing. Previous studies have shown that loss of ADAR1 editing activity leads to activation of the double-stranded RNA (dsRNA) immune response