Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit

Abstract

Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large subunit rRNAs and a number of ancillary "glue" proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.

Keywords: Trypanosoma cruzi; antitrypanosome drug design; biogenesis; multiply fragmented rRNA; ribosome structure.

Fig. 1.

Structure of the T. cruzi large subunit ribosome. (A) Cryo-EM map of the 60S subunit after sharpening, colored by local resolution and viewed from the subunit interface. (Left) Surface view. (Right) Central cut-away view. (B) Selected views of density for rRNA and proteins with associated ions and water molecule. (C) Some expansion segments in the unsharpened map of a large subunit, viewed from the solvent side. (C and D) rRNA architecture of the large subunit: interface (C) and solvent (D) view. (E) Some expansion segments in the unsharpened map of a large subunit, viewed from the solvent side.

Fig. 2.

Interactions stabilizing the srRNAs. (A) Scaffold formed by 5.8S rRNA, LSU-α, and LSU-β. PET, peptide exit tunnel. (B) srRNA2 (cyan). The components interacting with srRNA2 include the KSD stretch, H60, H64, H47, and uL3. (C) srRNA3 (forest green). The rRNA-contacting residues of the anchoring protein, eL6 and eL33, are shown. R94 and K97 are in the trypanosome-specific insertion of eL33. (D) srRNA4 (orange). The interacting residues are from the KSD stretch, H63, srRNA2, eL31, and uL3. (E) srRNA1 (magenta). W47 and H51 are from the trypanosome-specific insertion of eL34.

Fig. 3.

T. cruzi-specific expansion segments. (A) KSD contacts uL3, eL31, eL33, and eL14. (B) ES31L (yellow) in T. cruzi has the specific helix ES31L-h3 contacting eL8 and the 5′ end of 5.8S rRNA. ES31L-h1 and -h2 are only partially modeled. For comparison, ES31L from yeast is also shown here (in gray). (C) ES42L, emerging from H19, covers the cleavage sites of srRNA2–4 and blocks the extension of uL22.

Fig. 4.

A model of the proposed T. cruzi large subunit rRNAs assembly pathway. (A) 5.8S, LSU-α, and LSU-β form a scaffold. The gray color indicates the outline of the completely assembled large subunit. (B) srRNA2, -3, and ES7L are assembled. (C) srRNA4 and srRNA1 are assembled next. (D) 5S rRNA, ES42L, ES31L, and the KSD properly assemble to complete the large subunit. (E) Color key for the eight pieces of rRNA.