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

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. HighlightsO_LICryo-EM reveals the molecular architecture of the tetrameric Trypanosoma brucei cap-binding complex (TbCBC). C_LIO_LITbCBP110 is the kinetoplastid homolog of mammalian CBP80 and forms the scaffold for TbCBP20. C_LIO_LITbCBC has a bilobal architecture with TbCBP30 bridging the flexibly attached TbCBP66 subunit and the TbCBP20-TbCBP110 core complex. C_LIO_LITbCBC recognizes the m7G RNA cap independent of the other trypanosomatid-specific cap4 methylations. C_LIO_LIThe TbCBP66 subunit contains a binding site for dsRNA, augmenting the affinity of TbCBC for the SL RNA. C_LI