Proteomic Identification of Small-Subunit Ribosome Assembly Factors in Trypanosoma brucei

Trypanosomatid ribosomes display distinctive features, including extensive ribosomal RNA (rRNA) expansions and additional insertions in ribosomal proteins. Moreover, the region corresponding to the human 28S rRNA is fragmented into six molecules in these organisms with a duplication of the 3' fragment (ε) inLeishmania. Although these differences suggest that ribosome biogenesis in trypanosomatids may involve unique processing events, the molecular mechanisms underlying this process are still poorly characterized. In this study, we investigated the protein composition of pre-small-subunit (pre-SSU) complexes in Trypanosoma brucei. We generated cell lines expressing tagged versions of UTP6 and PNO1, two conserved ribosome biogenesis factors that provide complementary access to complexes of early SSU processome intermediates and later pre-40S maturation stages. Affinity purification followed by mass spectrometry identified numerous conserved ribosome biogenesis factors alongside a substantial set of trypanosomatid-specific proteins with no assigned function. Structural analyses revealed that many of these uncharacterized proteins contain predicted RNA-binding motifs or protein-protein interaction domains, and have been previously localized to the nucleolus, supporting potential roles in ribosome synthesis. Our findings expand the repertoire of candidate SSU assembly factors in kinetoplastids and highlight species-specific adaptations in ribosome biogenesis, providing a foundation for future functional studies targeting these unique components.