Uncommon RNA maturation: From RNA editing to cryptic splicing

Messenger RNA functionality depends on various maturation steps that not only enhance its stability but also add another layer of regulation. RNA splicing and editing contribute to functional diversification of proteins encoded by single genes. Previous and current research primarily focus on sequencing of bulk material and bioinformatics to identify editing and splice sites, but these methods inherently overlook the role of individual cells and different cell types. While developing an editing inducible system, evidence was provided to elucidate why C-to-U RNA editing and splicing were mutually exclusive. This led to the introduction of a novel molecular tool for the analysis of RNA splicing at single cell resolution with fluorescence readout. Its application has shown that the occurrence of non-canonical splice site usage might be not that rare and it identified consensus sequence motifs critical for effective cryptic RNA splicing. Additionally, the utilization of non-canonical splice donors supported the hypothesis of a wobble base at the second intronic position. Consistently, splicing of a natural intron appeared even without the influence of the different splice donors, indicating that intronic splicing regulatory elements have a significant impact on non-canonical splicing. Mitigation of these effects by integration of an exon in the sensor demonstrated that donor strength alone is sufficient for canonical splicing, whereas non-canonical splicing requires additional intronic sequence motifs. Mutations in the donors highlighted a specific position as critical for cryptic splicing. Cellular heterogeneity of non-canonical splice site usage underscores the context specific regulation of uncommon RNA processing events. Finally, this construct offers a promising tool for future research on splicing, potentially uncovering regulatory elements that affect non-canonical splicing. In parallel, the differentially spliced and edited human glycine receptor (GlyR) was investigated to assess its reaction to alkali metal ions. The GlyR is a widely distributed, ligand-gated, chloride-permeable ion channel in the central nervous system and its dysregulation is associated with various neurological disorders. Whole-cell voltage clamp electrophysiology experiments were conducted with HEK293T cells expressing homopentameric α2 and α3 GlyR variants. Cesium was established as an agonist of the GlyRs, exhibiting activation in a concentration- and editing-dependent manner. Notable differences in subunit-specific receptor responses emerged, such as the edited GlyR α2B´s reduced efficiency yet higher sensitivity to cesium. Additionally, atomistic molecular dynamic simulations identified two potential cesium interaction domains located near the glycine binding pocket and adjacent to the RNA edited site. This latter site involves amino acids D141, E192, and D194, which were subsequently mutated to encode for alanine or lysine. Patch clamp results indicated that D141 had no considerable impact on GlyR currents, E192K prolonged receptor desensitization and D194 was found to be crucial for general GlyR activation. Importantly, these effects were not due to differentially reduced cell surface expression among the analyzed GlyR mutants.