2023, Liashkovich, Ivan, Stefanello, Sílvio Terra, Vidyadharan, Reshma, Haufe, Günter, Erofeev, Alexander, Gorelkin, Peter V., Kolmogorov, Vasilii, Mizdal, Caren Rigon, Kouzel, Ian U., Shahin, Victor

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

2023, Barquin, Miguel, Kouzel, Ian U., Ehrmann, Beat, Basler, Michael, Gruber, Andreas J.

The usage of alternative terminal exons results in messenger RNA (mRNA) isoforms that differ in their 3′ untranslated regions (3′ UTRs) and often also in their protein-coding sequences. Alternative 3′ UTRs contain different sets of cis-regulatory elements known to regulate mRNA stability, translation and localization, all of which are vital to cell identity and function. In previous work, we revealed that ∼25 percent of the experimentally observed RNA 3′ ends are located within regions currently annotated as intronic, indicating that many 3′ end isoforms remain to be uncovered. Also, the inclusion of not yet annotated terminal exons is more tissue specific compared to the already annotated ones. Here, we present the single cell-based Terminal Exon Annotation database (scTEA-db, www.scTEA-db.org) that provides the community with 12 063 so far not yet annotated terminal exons and associated transcript isoforms identified by analysing 53 069 publicly available single cell transcriptomes. Our scTEA-db web portal offers an array of features to find and explore novel terminal exons belonging to 5538 human genes, 110 of which are known cancer drivers. In summary, scTEA-db provides the foundation for studying the biological role of large numbers of so far not annotated terminal exon isoforms in cell identity and function.