Parasite RNA-Binding Protein TbRRM1 Reveals Its Interaction Network and Binding Platform

Scientists have charted a wider circle of molecular partners around TbRRM1, an essential RNA-binding protein of the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis, known as sleeping sickness in humans and Nagana in animals. In a study combining affinity purification, mass spectrometry and computational structural modeling, researchers tagged the endogenous TbRRM1 protein in procyclic parasite cells, pulled down its associated proteins, and then used protein-protein docking to predict how these partners might bind. The work identified candidate interactors linked to RNA metabolism, translation and chromatin organization, and singled out an ATP-dependent RNA helicase as the strongest predicted binder, with a specific region of TbRRM1 emerging as the main interaction platform.

TbRRM1 is a serine-arginine-related RNA-binding protein carrying three RNA-recognition motifs, two C-terminal zinc finger domains and a nuclear localization signal, which gives it a predominantly nuclear distribution under normal conditions. In kinetoplastids such as T. brucei, protein-coding genes sit in polycistronic transcription units and are processed into individual mRNAs through trans-splicing and polyadenylation, so gene regulation is dominated by post-transcriptional mechanisms controlling mRNA maturation, export, stability and translation. RNA-binding proteins, including large families of RNA-recognition motif proteins, are central to these processes, and TbRRM1 is essential in both procyclic and bloodstream forms of the parasite, making it an attractive target for antiparasitic drug discovery.

Earlier work had already sketched part of the picture. Transcriptome-wide RNA immunoprecipitation in procyclic cells showed that TbRRM1 selectively associates with subsets of mRNAs, enriched for long coding-region transcripts and many stage-regulated messages more abundant in bloodstream forms. Global splice site selection is largely unaffected when TbRRM1 is depleted, indicating it is not a general trans-splicing factor. Instead, TbRRM1 links nuclear RNA binding with chromatin organization: its depletion increases nucleosome occupancy at defined genomic loci and produces visible chromatin compaction, reduces global gene transcription, and the protein interacts with multiple core histones. TbRRM1 also redistributes from nucleus to cytoplasm upon heat shock, with nuclear reaccumulation during recovery, and has been detected along a polycistronic transcription unit on chromosome 9, preferentially occupying RNA polymerase II-transcribed regions. Known interactors include DRBD4/PTB2, retrotransposon hotspot proteins, TRRM2 and nucleolar RNA helicase II, several of these interactions being RNA-independent, plus associations with ZC3H28 and the export factor TbMex67.

To find additional partners, the team built a functionally complementary system. They used a procyclic cell line in which TbRRM1 can be silenced with tetracycline-induced RNAi targeting the 5′ untranslated region, and transfected it with a construct that added 6xHis-3xFLAG epitopes to the N-terminus of one endogenous TbRRM1 allele. Because the RNAi targets the native 5’UTR while the tagged allele’s 5’UTR was replaced with the aldolase 5’UTR, induction of silencing knocks down only the untagged allele, leaving the tagged protein untouched. Western blotting confirmed that in the tagged line, tetracycline addition eliminated the wild-type band while the higher-molecular-weight FLAG-tagged band persisted, and immunofluorescence showed the tagged protein retained the same subcellular localization as the wild-type version.

Functionality was then verified through rescue. In the parental RNAi line, tetracycline induction caused a growth defect consistent with the lethal phenotype of TbRRM1 depletion. In the endogenously tagged clone, induced and non-induced cultures grew at comparable rates over 72 hours of daily counting, demonstrating that the N-terminally tagged TbRRM1 fully rescued the lethal phenotype and retained proper localization and biological activity. This validation was critical, because it supports using the tagged allele for interaction studies: the fusion protein evidently preserves the key molecular interactions required for essential activity, so proteins co-purifying with it likely reflect genuine associations rather than artifacts of a disabled bait.

With the validated system in hand, the researchers performed pull-down assays from lysates of about 1.9 billion tetracycline-induced tagged parasites using anti-FLAG agarose beads, running the identical procedure on untagged parental parasites as a negative control. Eluted proteins were analyzed by liquid chromatography-tandem mass spectrometry at a university facility, with high-confidence peptide filtering at a 1% false discovery rate. The comparison identified 13 proteins with high confidence scores, one of which, a universal minicircle sequence-binding protein, was excluded because it also appeared in the untagged control sample. The remaining candidates spanned functional categories: translation-associated proteins including elongation factor 1-alpha, a putative 60S ribosomal protein L9 and a putative mitochondrial LSU ribosomal protein; chromatin-associated core histones H2A/H2B/H3/H4; RNA metabolism proteins including the ATP-dependent RNA helicase and the previously known partner DRBD4/PTB2; the ZFP family member ZC3H5; an atypical variant surface glycoprotein; alpha-tubulin; and several conserved hypothetical proteins.

The team then turned to structural modeling and docking to test whether these associations are physically plausible. They divided full-length TbRRM1 into its defined domains, RRM1, RRM2, RRM3 and a zinc finger pair, plus an extended construct spanning RRM3 together with both zinc fingers, and predicted three-dimensional structures with AlphaFold3, retaining models with predicted template modeling scores of at least 0.6. Protein-protein docking used a Fourier transform-based algorithm, ranking poses by an energy score combining van der Waals, electrostatic and solvation terms, with stability criteria requiring the lowest-energy pose below minus 10 kilocalories per mole and an energy gap of at least 5 kilocalories per mole over the next pose. Binding free energies were estimated with PRODIGY and buried surface areas with FreeSASA. All tested interactors showed favorable interaction energies with at least one TbRRM1 domain. The ATP-dependent RNA helicase stood out, with docking energies consistently below minus 31 kilocalories per mole across all domain-interactor combinations, the highest mean energy across the four domains, and top rankings by both predicted binding free energy and buried surface area in three of the four domains, a pattern the authors report as statistically significant by an exact binomial test.

The predicted binding free energies for the best complexes fell below minus 8 kilocalories per mole for all candidates, and buried surface areas approached or exceeded 1000 square angstroms, which the researchers interpret as favorable interaction profiles. Five candidates, namely DRBD4/PTB2, ZC3H5, RPL9, one hypothetical protein and alpha-tubulin, ranked as the next most favorable partners after the helicase by mean binding free energy across the four domains, ranging from minus 7.7 to minus 9.5 kilocalories per mole. Notably, STRING network analysis revealed no previously known or predicted associations between TbRRM1 and these interactors in T. brucei, underscoring how little is annotated about this parasite’s interactome and the value of combining proteomics with bioinformatic validation. The detection of the known partner DRBD4/PTB2 alongside novel candidates supports the robustness of the approach, while the authors caution that differences from earlier datasets may stem from technical factors such as the liquid chromatography-based workflow, the affinity tag, sample preparation and the complementation system itself.

To probe the helicase interaction in detail, the researchers docked it against the extended RRM3-zinc finger construct and three mutant versions carrying single or double amino acid substitutions. The mutations destabilized the predicted complexes in a strikingly non-uniform way. A double substitution in RRM3 shifted the docking energy from minus 39.75 to minus 20.8 kilocalories per mole and raised the predicted binding free energy by 3.99 kilocalories per mole, while a histidine-to-alanine change in the second zinc finger raised it by 2.60 kilocalories per mole; a corresponding change in the first zinc finger had minimal effect, shifting binding free energy by only 0.29 kilocalories per mole. Buried surface areas told the same story, falling from 2210.70 square angstroms in the wild-type complex to 1177.11, 2055.53 and 1606.08 square angstroms for the three mutants, decreases of roughly 46, 7 and 27 percent. Residue-level analysis showed the wild-type interface engaged 32 residues, of which the mildest mutant retained 13 of 24, while the other two mutants retained only five and two, and the mutated histidines themselves vanished from their respective interfaces. Most strikingly, the RRM3 double mutant’s interface was essentially non-overlapping with the wild-type reference, consistent with the fragment repositioning to an entirely different region of the helicase surface, even though neither substituted residue contacts the helicase directly, implying the substitutions reshape the interface indirectly through structural effects on domain folding.

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. The stress connection is reinforced by the localization experiments: under control conditions TbRRM1 was mainly nuclear, but heat shock drove clear cytoplasmic redistribution, and inhibition of transcription or trans-splicing with actinomycin D or sinefungin produced partial relocalization to both compartments, echoing stress-induced relocalization reported for its T. cruzi ortholog and the formation of cytoplasmic stress granules in T. brucei during heat shock. The authors also frame TbRRM1 as a potential druggable target, noting that its T. cruzi ortholog can be targeted by small molecules, with sorafenib tosylate showing trypanocidal activity and evidence suggesting ortholog inhibition may contribute to that effect. The RRM3-zinc finger region identified as the major interaction platform represents a potential site for future structure-based drug discovery, though further experimental work will be needed to determine whether this region can be pharmacologically targeted and whether disrupting TbRRM1 interactions compromises parasite viability. All interaction findings for the novel partners remain candidate associations backed by computational prediction, and the docking and mutational results are in silico estimates rather than experimentally measured binding data.

Subject of Research: Mapping the interaction partners and predicted binding interfaces of the essential RNA-binding protein TbRRM1 in Trypanosoma brucei.

Article Title: Mapping TbRRM1 protein interactions and predicting binding interfaces in Trypanosoma brucei

Article References: Corso, M. C., Níttolo, A. G., Benacerraf, A. L., Cardozo, T., De Gaudenzi, J. G., & Levy, G. V. (2026). Mapping TbRRM1 protein interactions and predicting binding interfaces in Trypanosoma brucei. Frontiers in Chemical Biology, 5, Article 1957286. https://doi.org/10.3389/fchbi.2026.1957286

Image Credits: AI Generated

DOI: 10.3389/fchbi.2026.1957286

Keywords: Trypanosoma brucei, TbRRM1, RNA-binding protein, protein-protein interaction, mass spectrometry, molecular docking, RNA helicase, zinc finger domain, sleeping sickness, post-transcriptional regulation, proteomics, chromatin organization