Project Area A: "Modifications look for Processing"

The projects in Area A are characterized by long-standing expertise in RNA modification, and correspondingly, strong published or preliminary data that provide a solid base for exploring connections to processing events. In certain projects, the processing event is already characterized to a degree that warrants investigating mechanistic and functional effects.

Area A, provides central competences in RNA modification through numerous PIs that are firmly established in the field. Paramount among these is competence in modification analytics in A01, A02 and A06, documented by numerous joint publications, e.g., Frye, Tuorto, Stoecklin, Helm, & Lyko, Jäschke & Helm . Further internal connectivity within Area A stems from the fact that A03, A04 and A05 are connected by immunology as a common topic. Documentation of effective prior collaboration are several joint publications e.g. Ruggieri & Stoecklin, Helm & Butter and Dalpke & Helm , (see Publications and existing networks). Several PIs participate in joint DFG group funding instruments, e.g. TRR 186 (Ruggieri, Stoecklin) or SPP1784 (Dalpke, Helm, Jäschke, Lyko, Papavasiliou). The above connections solidly testify to a sound pre-established collaboration network that will provide a starting boost to RMaP.

A01

Mechanistic Insights into tRNA Guanosine Modifications m¹G and m⁷G

A01 investigates the role of tRNA guanosine modifications (m¹G and m⁷G), formed by METTL1 and TRMT5, in regulating mRNA translation in cancer cells. By integrating advanced chemical biology, proteomics, and both in vitro and in vivo assays, the project will unravel the catalytic and non-catalytic functions of these enzymes in RNA processing and protein synthesis. Ultimately, the study aims to reveal how these tRNA modifications contribute to tumor growth and treatment resistance.

A02

The biological relevance of NAD⁺-capping of RNA

Non-canonical RNA caps like NAD exist in both prokaryotes and eukaryotes, but their broader biological roles remain unclear. To address this issue, we will: (1) enhance the HELIOS NAD-seq platform for dynamic profiling of NAD-capped RNAs; (2) examine the processing of the NAD transcriptome in human cells using pharmacological and genetic tools; and (3) develop visualization methods employing aptamers and NAD-binding proteins to study subcellular localization. These efforts will elucidate NAD-RNA dynamics and the role of NAD capping in RNA modification and processing across various models.

A03

How RNA modifications affect RNA processing and TLR8 stimulation in innate immune cells

Toll-like receptors (TLR) 7 and 8 recognize microbial RNA thereby activating an innate immune response. The Dalpke group identified a role for RNase6 and 2’-O-methyl modifications in activation of TLR8. With the expertise of the Höbartner group we will now employ a chemistry driven approach to generate RNA modifications to study their immune effects in a structure-activity approach. Furthermore we will investigate regulation of bacterial RNA modifications under stress as mean of immune evasion and quantitatively analyze ribosomal RNA modifications with respect to foreign/self discrimination.

A04

Ensemble analysis of mRNA editing, modification and processing in macrophages

Macrophages are important cells of the innate immune system and require modification of adenosine in messenger RNA (mRNA) to fulfill their functions. Project A04 interrogates the interplay and interdependency between adenosine to inosine deamination and N⁶-adenosine methylation in the mouse macrophage cell line RAW264.7. We focus on the effect of these modifications on nuclear mRNA processing including splicing and polyadenylation site selection, as well as their role in transcription-dependent control of mRNA turnover.

A05

Dynamics of modifications and processing of RNAs in the flavivirus replication cycle

Project A5 aims to develop purification methods to study the RNA modifications that occur in the dengue virus (DENV) genome (gRNA) during the viral life cycle and their relevance. During FP1, we identified a single m⁵C site in DENV gRNA and uncovered its role in promoting gRNA turnover. In FP2, we will investigate the mechanistic regulation of this modification, including potential reader and eraser proteins, and investigate conservation in other DENV serotypes. We will further refine an in-gel hybridization technique for the isolation of sufficient quantities of short DENV subgenomic sfRNA for modification analysis by LC-MS.

A06

Interplay between tRNA processing and tRNA modifications

The maturation of transfer RNAs (tRNAs) involves the intricate interplay between processing and modification steps. We aim to dissect the molecular mechanisms that coordinate these events during tRNA biosynthesis. To this end, we will map the modification status of pre-tRNAs at distinct maturation stages. Moreover, we will elucidate the hierarchical interdependencies and structures of key enzyme complexes, their subcellular localization, and the biological roles of tRNA introns.

A07

Uncovering the role of m¹A methylation in mitochondrial mRNA stability, translation, and function

Mitochondrial RNA modifications, including N1-methyladenosine (m¹A), are thought to regulate mitochondrial (mt) translation, but their biological effects are not well understood. Here, the impact and mechanism of m¹A on mt-mRNA processes such as processing, turnover, decay, translation, ribosome stalling, and double-stranded mt-mRNA formation will be addressed. A reliable method using nanopore technology will be developed to map and quantify mitochondrial modifications. Finally, the m¹A mt-mRNA landscape will be examined under normal, stress, and disease conditions

A08

How VIRMA Shapes m⁶A-Driven Transcript Modifications

The modification of mRNA generating N6-methyladenosine (m⁶A) is catalyzed by METTL3–METTL14 methyltransferase complex and depends on different cofactors. This project explores how the largest of these cofactors, VIRMA, orchestrates m⁶A deposition to regulate transcription, RNA processing and stability, promoting cancer cell survival. Using structural biology and transcriptome profiling, this study will uncover how cancer-associated VIRMA mutations alter m⁶A modification profiles and influence tumor progression.