Functional and protein interaction analysis of Nop7 in trypanosomatid parasites

Nucleolar protein 7 (Nop7), also known as Pescadillo or PES1, is a conserved factor required for ribosome biogenesis, cell proliferation, and nucleolar organization in opisthokonts; however, its function in early-branching eukaryotes remains poorly understood. Here, we characterize Nop7 in the kinetoplastid parasites Trypanosoma brucei and Leishmania major, organisms that possess atypical ribosomes containing extensively fragmented 28S-type ribosomal RNA. Structural in silico analyses revealed lineage-specific features in trypanosomatid Nop7 proteins that may support specialized protein-protein interactions. Using tandem affinity purification coupled with mass spectrometry, we defined the Nop7-associated interactome, identifying numerous conserved trans-acting ribosome assembly factors, including Erb1 and Ytm1, consistent with the presence of a PeBoW-like complex involved in 60S ribosome subunit maturation. Comparative proteomic analyses also uncovered several previously uncharacterized nucleolar proteins, suggesting the existence of parasite-specific components within the ribosome biogenesis machinery. Functional assays showed that RNAi-mediated depletion of TbNop7 causes severe growth defects, morphological abnormalities, and significant alterations in 5.8S and 28S pre-rRNA maturation. Collectively, these findings provide the first proteomic map of the Nop7 interaction network in trypanosomatids and offer new insights into ribosome biogenesis in evolutionarily divergent eukaryotes. SIGNIFICANCE: Trypanosoma brucei and Leishmania major are two medically relevant kinetoplastid parasites that possess an atypical cytosolic ribosome, whose large subunit is composed of eight ribosomal RNA species of distinct lengths and origins, suggesting specialized biogenesis mechanisms. Here, we characterize the nucleolar protein 7 (Nop7) from T. brucei and L. major and demonstrate that Nop7 is necessary for parasite proliferation, cell cycle progression, and ribosome biogenesis. Bioinformatics analyses reveal conserved functional domains, predicted SUMOylation sites, and trypanosomatid-specific structural features that may facilitate processing of their unique 28S-like rRNA. Proteomic interaction networks indicate that Nop7 associates with canonical ribosome biogenesis factors as well as parasite-specific partners, highlighting an evolutionarily adapted molecular machinery. Collectively, our findings provide new insights into ribosome biogenesis in early-diverging eukaryotes and suggest some potential molecular targets for antiparasitic therapy.