Trypanosomatid parasites, including different trypanosoma and leishmania species, are responsible for severe human and animal diseases of medical and economic importance worldwide. The Kowalinski group has revealed the architecture and function of an RNA-binding complex that is key to mRNA biogenesis in these parasites [1].
Like human mRNAs, all trypanosome mRNAs are capped with a chemical modification at the 5’-end. In humans, this cap is generated co-transcriptionally when the mRNA emerges from the RNA polymerase II. The pre-mRNA cap is rapidly bound by the nuclear cap-binding complex (CBC). CBC is an important platform for quality control factors interacting with the newly synthesised mRNA to ensure its correct localisation and determine its fate [2]. In trypanosomes, the mRNA cap is hypermodified with seven methylations (termed cap4) instead of one or two in humans [2]. However, the trypanosome nuclear cap-binding complex (TbCBC) consists of four subunits, instead of two in humans or yeast. For three TbCBC subunits, no function could previously be assigned based on sequence homology or AlphaFold models [4].
Using cryo-EM the Kowalinski group showed that TbCBC consists of a cap-binding core formed by the TbCBP20 and TbCBP110 subunits, which resembles the human complex. A combination of AlphaFold predictions, small-angle scattering (SAXS) and co-precipitation assays reveals that the TbCBP30 subunit acts as a flexible connector between the cap-binding core and the TbCBP66 subunit. In the next step, biochemical binding assays with RNAs carrying different cap structures were used to assess the RNA-binding properties of TbCBC. A “normal” m7G cap was required and sufficient for interaction with the cap-binding core; the additional trypanosome-specific cap4 modifications were unnecessary. This agreed with observations from the cryo-EM structure since specific interactions of TbCBC with the trypanosome-specific methylations could not be detected. Moreover, the group discovered that the fourth subunit, TbCBP66, provides a second RNA-binding site, specifically recognising double-stranded RNA, assigning a novel function to TbCBP66. This secondary anchor point increases the affinity of TbCBC for the Spliced Leader RNA (SL RNA), a short stretch of RNA ligated to all 5’ mRNA ends in trypanosomes.
In conclusion, by leveraging many PSB platforms (EEF, biophysics, BM29 and CM01) the results generated exemplify how trypanosomatids have altered a conserved molecular machinery to adapt it to their unique RNA metabolism. TbCBC is vital for parasite-specific obligatory RNA processing through trans-splicing. In trans-splicing, primary polycistronic pre-mRNAs are processed into mature individual translatable mRNAs by the addition of a specialised 5’ RNA sequence. Understanding TbCBC paves the way for revealing how the trans-splicing mechanism works – a research line for which Eva Kowalinski was recently awarded a prestigious ERC Consolidator grant (see announcements). Additionally, in the future, anti-parasite therapies may target TbCBC, which is conserved within multiple trypanosomatid species, a project for which the Kowalinski lab has recently teamed up with the Hart lab at the IBS through a ‘Grenoble Attractiveness and Excellences Initiative‘ funded by the France 2030 framework of the French National Research Agency (ANR). Disrupting trypanosome RNA processing is expected to open new avenues for treatment, with potential benefits for human health and the economy.
H. Bernhard (EMBL / IBS), H. Petrzilkova (EMBL), L. Dolce(EMBL / IAB) and E. Kowalinski (EMBL)
[1] Bernhard H, Petrzilkova H, Popelarova B, Ziemkiewicz K, et al. (2024) Nat Commun., 16, 685.
[2] Gonatopoulos-Pournatzis T, Cowling VH (2014) Biochem J., 457, 231-242.
[3] Agabian N (1990) Cell 61, 1157–1160.
[4] Li H, Tschudi C (2005) Mol Cell Biol., 25, 2216–2226.
Figure: Architecture of the Trypanosoma brucei nuclear cap-binding complex (TbCBC). Hybrid structural model combining cryo-EM, SAXS and biochemical data.