Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex

Fig. 1. Cryo-EM structures of the T. brucei cap-binding complex. A Chemical structure of the 5’-end of the SL RNA with the cap4 modifications and m7G moiety indicated. B Domain organization of the TbCBC subunits. TbCBP110 (green), TbCBP20 (yellow), TbCBP30 (pink) and TbCBP66 (blue). Structural information for TbCBP110 and TbCBP20 is based on cryo-EM data from this study. The domain organization of TbCBP66 and the structural elements of TbCBP30 are based on Alphafold2 structure predictions. Interactions are indicated in gray. Key functional residues are indicated. C Size exclusion chromatography profiles of different CBC complexes that could be obtained through co-expression: TbCBC-tetramer (containing TbCBP20, TbCBP110, TbCBP30, TbCBP66), TbCBC-trimer (containing TbCBP20, TbCBP110, TbCBP30), TbCBC-dimer (containing TbCBP20, TbCBP110). A Superdex 200 Increase 3.2/300 column was used, the data is representative for two or more runs. D Cap-binding assay on immobilized γ-Aminophenyl-m7GTP resin with the TbCBC-tetramer and addition of different putative competitor molecules. I = input, P = pull-down. A Coomassie-stained 12% SDS-PAGE is shown as a representative gel of three repetitions of the…

Fig. 2. The structure of T. brucei CBP110. A T. brucei CBP110 is an α-helical protein with three consecutive domains: N-terminal (NTD), middle MIF4G-like, and C-terminal (CTD) domain. Cartoon representation with tubes representing α-helices. View similar to Fig. 1E, TbCBP20, and TbCBP30 models removed for clarity. TbCBP20 would be positioned in front of the view. B Human CBP80 (PDB: 1h2t35, with its three MIF4G-like domains has a similar shape and domain arrangement. C Comparison of TbCBP110 NTD and human CBP80 MIF4G-1 in topological coloring (N-terminus of the domain in blue to C-terminus of the domain in red); cartoon model of the domains and topological schematic. D Comparison of TbCBP110 CTD and human CBP80 MIF4G-3, as in (C). E Superposition of the TbCBP110 MIF4G-like domain (green) and human CBP80 MIF4G2 (blue). The domains superpose with an RMSD of 1.095 Å across 59 pruned atom pairs.

Fig. 3. RNA cap coordination in T. brucei CBP20. A Cartoon representation of TbCBP20 of the TbCBC-trimer-cap4 sample in shades of yellow indicating the three domains of TbCBP20. The TbCBP30 peptide bound to the CTD of TbCBP20 is also shown, with contact residues in stick representation. The cap-ligand is shown as a stick model, and its flexibility is indicated. B Residues in the m7GTP binding pocket of T. brucei and (C) human CBP20, based on PDB:1H2T35. D Deep-EMhancer post-processed trimer cryo-EM map transparent with cartoon model and cap4-hexanucleotide in stick representation and at level 0.3 and (E) at a very reduced level of 0.03. F Electrophoresis mobility shift assay (EMSA) of TbCBC-dimer binding to the SL RNA exon with different 5’ chemistry. G EMSA of TbCBC-trimer binding to the SL RNA exon with different chemistry. EMSAs in (F) and (G) were run in triplicate reactions; the intensity of PAGE gel bands was normalized in relation to the protein-free RNA control band. A Hill model was used to fit the binding curve. Source data are provided as a Source Data file.

Fig. 4. TbCBP30 is the bridging subunit between the TbCBP20-TbCBP110 core and TbCBP66. A DeepEMhancer post-processed map of the TbCBC-trimer-cap4 sample is shown at level 0.3. Colors as in previous figures. B A hydrophobic helix of TbCBP30 (in cartoon representation) binds to a hydrophobic groove on TbCBP110 (surface representation with ChimeraX hydrophobicity coloring)90. C Co-precipitation assay of co-expressed HA-tagged TbCBP20, TbCBP110, TbCBP66 with mCherry-TbCBP30 mutants and Western blot detection. A representative Western blot of more than three experiments is shown. D Binding of N-terminal portions of the TbCBP30 peptide to TbCBP20 and into the groove between TbCBP20 and TbCBP110. TbCBP20 and TbCBP110 are transparent surface representations with TbCBP30-facing residues as cartoons and sticks. TbCBP30 in cartoon representation, with interaction residues as sticks. E Fluorescence data of a microscale thermophoresis experiment determining the interaction between the TbCBC-dimer and TAMRA-labeled CBP30(R34-G60) (n = 3). F Co-precipitation assay of TbCBP66 with mCherry-CBP30 truncations western blot detection. A representative western blot of more than three experiments is shown.…

Fig. 5. T. brucei CBP66 is the dsRNA-binding subunit of TbCBC. A EMSA of TbCBC-tetramer binding to the SL RNA exons with different 5’ chemistry. B Size exclusion chromatography of the engineered TbCBP66* (CBP30(G141-N220)-CBP66 fusion), a representative curve of more than 3 experiments is shown. C EMSA probing the interaction of TbCBP66* with different uncapped RNAs: the 39 nt SL RNA exon (same ligand as in Fig. 3F, G), a 15 nt single-stranded RNA (A1-U15 of the SL RNA), and the 25 nt SL RNA hairpin loop comprising A14-G39 of the SL RNA. EMSAs in (A) and (C) were run in triplicate reactions, and the intensity of PAGE gel bands was normalized in relation to the protein-free RNA control band; A Hill model was used to fit the binding curve. D The Alphafold2 model of TbCBP66 indicates three domains with exposed positive charges on all domains. A surface representation of the Alphafold2 model and the Predicted aligned error (PAE) plot indicating the domain position confidence are shown. E Fluorescence polarization (FP) assay probing the binding of the 39 nt SL RNA exon to TbCBP66* single point mutants. The assay was run in triplicate and fit with a nonlinear sigmoidal fit model. See also…

Fig. 6. Model of the tetrameric kinetoplastid cap-binding complex with SL RNA. Colors as in Fig. 1B.