Mapping TbRRM1 protein interactions and predicting binding interfaces in Trypanosoma brucei

Introduction TbRRM1 is an essential serine‐arginine-related RNA‐binding protein containing three RNA‐recognition motifs (RRM) and two C-terminal zinc finger (ZF) domains, implicated in RNA metabolism, chromatin organization and transcriptional regulation in Trypanosoma brucei . Although previous studies identified TbRRM1‐associated factors, its broader interaction network and the structural basis of these associations remain an area of ongoing investigation. Methods Here, we combined endogenous tagging, functional complementation assays, affinity purification coupled to LC‐MS/MS and in silico structural analyses to characterize novel TbRRM1 interacting partners. Results Rescue experiments demonstrated that the N-terminally tagged TbRRM1 protein fully reverted the lethal phenotype caused by depletion of the endogenous allele, confirming that the tagged protein retained proper subcellular localization and biological activity. Pulldown assays using the endogenously tagged line identified multiple candidate interactors associated with RNA metabolism, translation and chromatin organization, including an ATP-dependent RNA helicase, the known TbRRM1-partner double RNA-binding domain 4 (DRBD4/PTB2) and the elongation factor 1-alpha, among others. Computational docking analyses combined with binding free energy and buried surface area calculations supported favorable interactions between TbRRM1 and all candidate proteins, highlighting the ATP-dependent RNA helicase as displaying the strongest and most consistent interaction profile. Domain-mapping analyses identified a region encompassing the third RRM and both ZF domains as the major interaction platform. In silico mutational analyses further demonstrated that specific substitutions within this region markedly reduced predicted binding affinity and altered docking interfaces. Discussion Together, these findings expand the functional network of TbRRM1 and support its role as a dynamic regulator connecting RNA metabolism, stress responses and translation-associated pathways in T. brucei .