Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5'-end of each mRNA. The 5'-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.

Centre of New Technologies University of Warsaw Warsaw Poland

Department of Experimental Biology Section of Microbiology Faculty of Science Masaryk University Brno Czech Republic

Division of Biophysics Institute of Experimental Physics Faculty of Physics University of Warsaw Warsaw Poland

EMBL Grenoble 71 Avenue des Martyrs Grenoble France

Institut de Biologie Structurale Grenoble France

Institute for Advanced Biosciences INSERM U1209 CNRS UMR 5309 Université Grenoble Alpes Grenoble France

Institute of Organic Chemistry Center for Molecular Biosciences Innsbruck University of Innsbruck Innsbruck Austria

University of Bordeaux INSERM CNRS ARNA Laboratory U1212 UMR 5320 Bordeaux France

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Burza, S., Croft, S. L. & Boelaert, M. Leishmaniasis. Lancet392, 951–970 (2018). PubMed

Büscher, P., Cecchi, G., Jamonneau, V. & Priotto, G. Human African trypanosomiasis. Lancet390, 2397–2409 (2017). PubMed

Pérez-Molina, J. A. & Molina, I. Chagas disease. Lancet391, 82–94 (2018). PubMed

Ungogo, M. & de Koning, H. Drug resistance in animal trypanosomiases: Epidemiology, mechanisms and control strategies. Preprints at10.20944/preprints202401.1300.v1 (2024). PubMed PMC

Kramer, S. Nuclear mRNA maturation and mRNA export control: from trypanosomes to opisthokonts. Parasitology148, 1196–1218 (2021). PubMed PMC

Clayton, C. E. Gene expression in Kinetoplastids. Curr. Opin. Microbiol.32, 46–51 (2016). PubMed

Clayton, C. Regulation of gene expression in trypanosomatids: living with polycistronic transcription. Open Biol.9, 190072 (2019). PubMed PMC

Koch, H., Raabe, M., Urlaub, H., Bindereif, A. & Preußer, C. The polyadenylation complex of Trypanosoma brucei: Characterization of the functional poly(A) polymerase. RNA Biol.13, 221–231 (2016). PubMed PMC

Matthews, K. R., Tschudi, C. & Ullu, E. A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes. Genes Dev.8, 491–501 (1994). PubMed

Vassella, E., Braun, R. & Roditi, I. Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site: a dual role for polypyrimidine tracts in trypanosomes? Nucleic Acids Res.22, 1359–1364 (1994). PubMed PMC

Michaeli, S. Trans-splicing in trypanosomes: machinery and its impact on the parasite transcriptome. Future Microbiol.6, 459–474 (2011). PubMed

Agabian, N. Trans splicing of nuclear pre-mRNAs. Cell61, 1157–1160 (1990). PubMed

Sutton, R. E. & Boothroyd, J. C. Evidence for trans splicing in trypanosomes. Cell47, 527–535 (1986). PubMed PMC

Murphy, W. J., Watkins, K. P. & Agabian, N. Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell47, 517–525 (1986). PubMed

Gilinger, G. & Bellofatto, V. Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms. Nucleic Acids Res.29, 1556–1564 (2001). PubMed PMC

Harris, K. A. Jr, Crothers, D. M. & Ullu, E. In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations. RNA1, 351–362 (1995). PubMed PMC

Bruzik, J. P., Van Doren, K., Hirsh, D. & Steitz, J. A. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature335, 559–562 (1988). PubMed

Preußer, C., Jaé, N., Günzl, A. & Bindereif, A. in RNA Metabolism in Trypanosomes 49–77 (Springer Berlin Heidelberg, Berlin, Heidelberg, 2012).

Tkacz, I. D. et al. Analysis of spliceosomal proteins in Trypanosomatids reveals novel functions in mRNA Processing. J. Biol. Chem.285, 27982–27999 (2010). PubMed PMC

Mandelboim, M., Barth, S., Biton, M., Liang, X.-H. & Michaeli, S. Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis. J. Biol. Chem.278, 51469–51478 (2003). PubMed

Luz Ambrósio, D. et al. Spliceosomal proteomics in Trypanosoma brucei reveal new RNA splicing factors. Eukaryot. Cell8, 990–1000 (2009). PubMed PMC

Goncharov, I., Palfi, Z., Bindereif, A. & Michaeli, S. Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue. J. Biol. Chem.274, 12217–12221 (1999). PubMed

Mandelboim, M., Estrano, C. L., Tschudi, C., Ullu, E. & Michaeli, S. On the role of exon and intron sequences intrans-splicing utilization and cap 4 modification of the trypanosomatid Leptomonas collosoma SL RNA. J. Biol. Chem.277, 35210–35218 (2002). PubMed

Mair, G., Ullu, E. & Tschudi, C. Cotranscriptional cap 4 formation on the Trypanosoma brucei spliced leader RNA. J. Biol. Chem.275, 28994–28999 (2000). PubMed

Bangs, J. D., Crain, P. F., Hashizume, T., McCloskey, J. A. & Boothroyd, J. C. Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J. Biol. Chem.267, 9805–9815 (1992). PubMed

Perry, K. L., Watkins, K. P. & Agabian, N. Trypanosome mRNAs have unusual “cap 4” structures acquired by addition of a spliced leader. Proc. Natl. Acad. Sci. USA84, 8190–8194 (1987). PubMed PMC

Freistadt, M. S., Cross, G. A. & Robertson, H. D. Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5’ cap structure, m7GpppA*A*C(2’-O)mU*A. J. Biol. Chem.263, 15071–15075 (1988). PubMed

Ullu, E. & Tschudi, C. Trans splicing in trypanosomes requires methylation of the 5’ end of the spliced leader RNA. Proc. Natl. Acad. Sci. USA88, 10074–10078 (1991). PubMed PMC

Gonatopoulos-Pournatzis, T. & Cowling, V. H. Cap-binding complex (CBC). Biochem. J.457, 231–242 (2014). PubMed PMC

Müller-McNicoll, M. & Neugebauer, K. M. How cells get the message: dynamic assembly and function of mRNA–protein complexes. Nat. Rev. Genet.14, 275–287 (2013). PubMed

Rambout, X. & Maquat, L. E. The nuclear cap-binding complex as choreographer of gene transcription and pre-mRNA processing. Genes Dev.34, 1113–1127 (2020). PubMed PMC

Izaurralde, E. et al. A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell78, 657–668 (1994). PubMed

Ohno, M., Kataoka, N. & Shimura, Y. A nuclear cap binding protein from HeLa cells. Nucleic Acids Res.18, 6989–6995 (1990). PubMed PMC

Calero, G. et al. Structural basis of m7GpppG binding to the nuclear cap-binding protein complex. Nat. Struct. Biol.9, 912–917 (2002). PubMed

Mazza, C., Segref, A., Mattaj, I. W. & Cusack, S. Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex. EMBO J.21, 5548–5557 (2002). PubMed PMC

Izaurralde, E. et al. A cap-binding protein complex mediating U snRNA export. Nature376, 709–712 (1995). PubMed

Kataoka, N., Ohno, M., Moda, I. & Shimura, Y. Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein. Nucleic Acids Res.23, 3638–3641 (1995). PubMed PMC

Flaherty, S. M., Fortes, P., Izaurralde, E., Mattaj, I. W. & Gilmartin, G. M. Participation of the nuclear cap binding complex in pre-mRNA 3’ processing. Proc. Natl. Acad. Sci. USA94, 11893–11898 (1997). PubMed PMC

Narita, T. et al. NELF interacts with CBC and participates in 3’ end processing of replication-dependent histone mRNAs. Mol. Cell26, 349–365 (2007). PubMed

Cheng, H. et al. Human mRNA export machinery recruited to the 5’ end of mRNA. Cell127, 1389–1400 (2006). PubMed

Schulze, W. M. & Cusack, S. Structural basis for mutually exclusive co-transcriptional nuclear cap-binding complexes with either NELF-E or ARS2. Nat. Commun.8, 1302–1314 (2017). PubMed PMC

Schulze, W. M., Stein, F., Rettel, M., Nanao, M. & Cusack, S. Structural analysis of human ARS2 as a platform for co-transcriptional RNA sorting. Nat. Commun.9, 1701–1715 (2018). PubMed PMC

Dubiez, E. et al. Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex. Cell Rep.43, 113639 (2024). PubMed

Gruber, J. J. et al. Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation. Cell138, 328–339 (2009). PubMed PMC

Sabin, L. R. et al. Ars2 regulates both miRNA- and siRNA- dependent silencing and suppresses RNA virus infection in Drosophila. Cell138, 340–351 (2009). PubMed PMC

Ohno, M., Segref, A., Bachi, A., Wilm, M. & Mattaj, I. W. PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell101, 187–198 (2000). PubMed

Gebhardt, A. et al. mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3. Nat. Commun.6, 8192 (2015). PubMed PMC

Kiriyama, M., Kobayashi, Y., Saito, M., Ishikawa, F. & Yonehara, S. Interaction of FLASH with arsenite resistance protein 2 is involved in cell cycle progression at S phase. Mol. Cell. Biol.29, 4729–4741 (2009). PubMed PMC

Gromadzka, A. M., Steckelberg, A.-L., Singh, K. K., Hofmann, K. & Gehring, N. H. A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs. Nucleic Acids Res.44, 2348–2361 (2016). PubMed PMC

Viphakone, N. et al. Co-transcriptional loading of RNA export factors shapes the human transcriptome. Mol. Cell75, 310–323 (2019). PubMed PMC

Hosoda, N., Kim, Y. K., Lejeune, F. & Maquat, L. E. CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells. Nat. Struct. Mol. Biol.12, 893–901 (2005). PubMed

Lejeune, F., Ishigaki, Y., Li, X. & Maquat, L. E. The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. EMBO J.21, 3536–3545 (2002). PubMed PMC

Dantsuji, S., Ohno, M. & Taniguchi, I. The hnRNP C tetramer binds to CBC on mRNA and impedes PHAX recruitment for the classification of RNA polymerase II transcripts. Nucleic Acids Res.51, 1393–1408 (2023). PubMed PMC

Andersen, P. R. et al. The human cap-binding complex is functionally connected to the nuclear RNA exosome. Nat. Struct. Mol. Biol.20, 1367–1376 (2013). PubMed PMC

Lubas, M. et al. The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis. Cell Rep.10, 178–192 (2015). PubMed

Pabis, M., Neufeld, N., Shav-Tal, Y. & Neugebauer, K. M. Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20. Nucleus1, 412–421 (2010). PubMed PMC

Sato, H. & Maquat, L. E. Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin beta. Genes Dev.23, 2537–2550 (2009). PubMed PMC

Fortes, P. et al. The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation. Mol. Cell6, 191–196 (2000). PubMed

Dias, S. M. G., Wilson, K. F., Rojas, K. S., Ambrosio, A. L. B. & Cerione, R. A. The molecular basis for the regulation of the cap-binding complex by the importins. Nat. Struct. Mol. Biol.16, 930–937 (2009). PubMed PMC

Görlich, D. et al. Importin provides a link between nuclear protein import and U snRNA export. Cell87, 21–32 (1996). PubMed

Li, H. & Tschudi, C. Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei. Mol. Cell. Biol.25, 2216–2226 (2005). PubMed PMC

Mazza, C., Ohno, M., Segref, A., Mattaj, I. W. & Cusack, S. Crystal structure of the human nuclear cap binding complex. Mol. Cell8, 383–396 (2001). PubMed

Worch, R. et al. Diverse role of three tyrosines in binding of the RNA 5′ cap to the human nuclear cap binding. Complex. J. Mol. Biol.385, 618–627 (2009). PubMed

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature596, 583–589 (2021). PubMed PMC

Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods19, 679–682 (2022). PubMed PMC

Fawaz, M. V., Topper, M. E. & Firestine, S. M. The ATP-grasp enzymes. Bioorg. Chem.39, 185–191 (2011). PubMed PMC

McNally, K. P. & Agabian, N. Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo. Mol. Cell. Biol.12, 4844–4851 (1992). PubMed PMC

Badjatia, N., Ambrósio, D. L., Lee, J. H. & Günzl, A. Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1. Mol. Cell. Biol.33, 1965–1975 (2013). PubMed PMC

Gosavi, U., Srivastava, A., Badjatia, N. & Günzl, A. Rapid block of pre-mRNA splicing by chemical inhibition of analog-sensitive CRK9 in Trypanosoma brucei. Mol. Microbiol.113, 1225–1239 (2020). PubMed PMC

Apolit, C. et al. ABX464 (Obefazimod) Upregulates miR-124 to Reduce Proinflammatory Markers in Inflammatory Bowel Diseases. Clin. Transl. Gastroenterol.14, e00560 (2023). PubMed PMC

Betu Kumeso, V. K. et al. Efficacy and safety of acoziborole in patients with human African trypanosomiasis caused by Trypanosoma brucei gambiense: a multicentre, open-label, single-arm, phase 2/3 trial. Lancet Infect. Dis.23, 463–470 (2023). PubMed PMC

Begolo, D. et al. The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing. PLoS Pathog.14, e1007315 (2018). PubMed PMC

Shanmugasundram, A. et al. TriTrypDB: An integrated functional genomics resource for kinetoplastida. PLoS Negl. Trop. Dis.17, e0011058 (2023). PubMed PMC

Aslett, M. et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res.38, D457–D462 (2010). PubMed PMC

Weissmann, F. et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc. Natl. Acad. Sci. USA113, E2564–E2569 (2016). PubMed PMC

Pelosse, M. & Marcia, M. biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at theACTBlocus. Preprint at 10.1101/2024.04.18.590029 (2024).

Fitzgerald, D. J. et al. Protein complex expression by using multigene baculoviral vectors. Nat. Methods3, 1021–1032 (2006). PubMed

Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol.152, 36–51 (2005). PubMed

Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in -3.1. IUCrJ7, 253–267 (2020). PubMed PMC

Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods14, 331–332 (2017). PubMed PMC

Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol.192, 216–221 (2015). PubMed PMC

Tegunov, D. & Cramer, P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods16, 1146–1152 (2019). PubMed PMC

Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods14, 290–296 (2017). PubMed

Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol.180, 519–530 (2012). PubMed PMC

Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr.66, 486–501 (2010). PubMed PMC

Casañal, A., Lohkamp, B. & Emsley, P. Current developments in coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci.29, 1069–1078 (2020). PubMed PMC

Terwilliger, T. C., Adams, P. D., Afonine, P. V. & Sobolev, O. V. A fully automatic method yielding initial models from high-resolution cryo-electron microscopy maps. Nat. Methods15, 905–908 (2018). PubMed PMC

Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol.74, 531–544 (2018). PubMed PMC

Reolon, L. W. et al. Crystal structure of the Trypanosoma cruzi EIF4E5 translation factor homologue in complex with mRNA cap-4. Nucleic Acids Res.47, 5973–5987 (2019). PubMed PMC

Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci.30, 70–82 (2021). PubMed PMC

Manalastas-Cantos, K. et al. expanded functionality and new tools for small-angle scattering data analysis. J. Appl. Crystallogr.54, 343–355 (2021). PubMed PMC

Ziemkiewicz, K., Warminski, M., Wojcik, R., Kowalska, J. & Jemielity, J. Quick access to nucleobase-modified phosphoramidites for the synthesis of oligoribonucleotides containing post-transcriptional modifications and epitranscriptomic marks. J. Org. Chem.87, 10333–10348 (2022). PubMed PMC

Leiter, J., Reichert, D., Rentmeister, A. & Micura, R. Practical synthesis of cap‐4 RNA. ChemBioChem21, 265–271 (2020). PubMed PMC