Deciphering RNA modification patterns along single molecules: Modification crosstalk suppressing innate immunity

• Applicants: Dr. Janine Altmüller; Professor Dr. Christoph Dieterich
• Subject Area: General Genetics and Functional Genome Biology ; Bioinformatics and Theoretical Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548112559

Today, only a handful of mRNA modifications have been comprehensively mapped by different sequencing approaches. Thus, while there is reasonably good understanding on where modifications may occur in the transcriptome, quantitative and single molecule information is still lacking widely. Moreover, the interplay and cooccurrence of different modifications along individual molecules is largely missing except for well characterized tRNAs and rRNAs. However, to understand the regulatory code of RNA modifications on the immune system, but also on each other, such information is needed. Therefore, in this proposal we would like to address the following questions: 1) Are all molecules modified to the same extent? 2) Is there a crosstalk between different types of RNA modifications? 3) Is there a crosstalk between the same types of RNA modifications? 4) Is the deposition of modifications processive? 5) Is there a specific modification pattern that suppresses innate immune responses? We will address these questions by applying state-of-the-art sequencing technologies that allow long-read sequencing on RNAs that are either modified in vitro or in vivo. Using cells or animals of different genetic background will allow us to address the interplay of different RNA modifications but also the contribution of individual modifying enzymes to the site-specific introduction of modifications. Specifically, we will focus on the detection of the (co) occurrence and linear distribution of the abundant modifications m6A and Inosine. The spatiotemporal deposition of modifications along single molecules will help to decipher modification patterns that are critically required to suppress innate immune responses. Once such modification patterns have been identified, they can be artificially reintroduced in substrate RNAs and tested for their ability to interfere with immune activation. We seek to address the aforementioned open questions by employing complementary single molecule techniques such as DART-seq on the PacBio platform and direct RNA-seq on the Nanopore platform in combination with the relevant bioinformatic analysis approaches. A single molecule perspective allows us to characterize the occurrence and co-occurrence patterns of modifications in the entire mRNA transcriptome ensemble. RNA modification patterns identified here will provide the basis to understand the modification code that drives innate immune sensing of RNAs. This highly innovative proposal will establish for the first time the detection of multiple modifications along single molecules and define the coordination by which RNA modifications are inserted along molecules. The proposal brings together the expertise of experimental RNA biology, computational RNA biology and 3rd generation sequencing.

• DFG Programme: Research Grants
• International Connection: Austria
• Partner Organisation: Fonds zur Förderung der wissenschaftlichen Forschung (FWF)
• Cooperation Partner: Professor Dr. Michael F. Jantsch