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
DNMT2, NSUN2 and NSUN6 catalyze the transfer or a methyl group to the C5-position of specific cytosines in tRNA. The presence of 5-methylcytosine (m5C) can affect tRNA cleavage, and misregulation can lead to cellular differentiation defects and disease. By developing small-molecule modulators targeting the active-site cysteine, and a potent, selective RNA aptamers, the therapeutic potential of modulating methylase activity will be evaluated. These will also be used as tools, alongside genetic depletion, to investigate mechanisms by which m5C controls cell fate and differentiation.
A02
Modification at RNA 5’-ends with the non-canonical cap NAD+ has been described in bacteria, yeast and mammalian cell lines but the biological consequences of NAD+ capping remain poorly understood. Here, the influence of 5’-NAD+ capping on various processes in the life of an RNA, from its transcription, to RNA splicing, 3’-cleavage and polyadenylation, as well as RNA turnover will be addressed. Moreover, a possible influence of NAD+ capping on the introduction of further modifications, in particular methylations, into RNA will be assessed. Finally, the NAD+ decapping process will be investigated.
A03
Innate immune responses via endosomal Toll-like receptors (TLR) 7 and 8 are stimulated by exogenous microbial RNA. RNA-TLR interaction relies on RNA processing and is impeded by certain RNA modifications. Here, omics techniques will be used to study changes in subcellular proteomes, secretomes, and protein binding to immune-stimulatory RNA. The role of RNase 6 in microbial RNA processing, its cleavage mechanisms, and the role of inhibitory RNA 2’-O-ribose methylation will be analysed and further endosomal RNA interactors involved in RNA processing and TLR stimulation identified
A04
RNA modifications, including RNA editing by deamination, can be dynamic and occur not only in rRNA and tRNA, but also in mRNA. Catalogs of specific mRNA modifications and their effects on mRNA stability or translation efficiency have been assembled haphazardly from different cell lines. To obtain a comprehensive picture of complex interdependencies, here, a single cell line will be used to analyze connections between mRNA modification, degradation and translation, and characterize related RNA-binding proteins and RNA-associated complexes.
A05
RNA modifications in RNA viruses are linked to RNA processing, degradation, structure formation, and in particular, immune evasion. Despite their obvious importance, results on detection and mapping of viral RNA modifications are sketchy and in part controversial. Here, flaviviral genome RNA (gRNA) modification patterns upon infection of various host cells will be analysed in detail. Modification patterns will then be analyzed in a time-resolved manner, and their impact on gRNA processing, as well as on innate immune response will be assessed. Finally, comparisons will be drawn to other RNA viruses, including Sars-CoV-2.
A06
tRNAs are transcribed as precursors that require maturation, including non-conventional splicing and modification for function in translation. Here, modifications will be investigated with respect to tRNA processing, starting with reconstitution of enzymatic steps in vitro. Effects of unmodified/misprocessed tRNA will then be evaluated in vivo, as to how structural constraints establish a hierarchy in modification-processing interplay. Finally, modification patterns and fate of tRNAs upon ablation of introns, and formation of tRNA intronic circular RNAs will be characterized.
