Structural basis of Spliced Leader RNA recognition by the <i>Trypanosoma brucei</i> cap-binding complex

Abstract 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 ( Tb CBC) complex. We find that Tb CBC interacts with two distinct features of the SL RNA. The Tb CBP20 subunit interacts with the m 7 G cap while Tb CBP66 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 Tb CBC and the mammalian cap-binding complex will be crucial for considering the potential of Tb CBC as a target for anti-trypanosomatid drug development. Highlights Cryo-EM reveals the molecular architecture of the tetrameric Trypanosoma brucei cap-binding complex ( Tb CBC). Tb CBP110 is the kinetoplastid homolog of mammalian CBP80 and forms the scaffold for Tb CBP20. Tb CBC has a bilobal architecture with Tb CBP30 bridging the flexibly attached Tb CBP66 subunit and the Tb CBP20- Tb CBP110 core complex. Tb CBC recognizes the m 7 G RNA cap independent of the other trypanosomatid-specific cap4 methylations. The Tb CBP66 subunit contains a binding site for dsRNA, augmenting the affinity of Tb CBC for the SL RNA.