IDENTIFICATION OF RIBOSOME ASSEMBLY FACTORS INVOLVED IN SMALL SUBUNIT BIOGENESIS IN TRYPANOSOMA BRUCEI

Diseases caused by trypanosomatids, such as leishmaniasis and human trypanosomiasis, are neglected tropical diseases responsible for thousands of deaths and a significant economic burden. However, first-line treatments have remained unchanged for decades, highlighting the urgent need for novel compounds. A promising target for inhibitors is the ribosome—a complex composed of proteins and RNA responsible for protein translation, a universal process in living organisms. Trypanosomatids exhibit differences in ribosomal RNA structure and maturation compared to other eukaryotes, such as additional cleavage sites and large expansion segments, yet the identification of specific factors remains largely unexplored. The early stages of ribosome biogenesis have been poorly elucidated in trypanosomatids, despite being extensively characterized in humans and yeast. For this reason, we aim to identify and characterize protein factors involved in ribosome assembly and maturation in Trypanosoma brucei, specifically focusing on the biogenesis of the small ribosomal subunit. Through homologous recombination, we generated and cultured genetically modified cells containing molecular tags on the bait proteins UTP6 or PNO1, two key proteins involved in early and late stages of ribosome biogenesis. After obtaining suitable cell mass, we fractionated the cell extracts using affinity chromatography and analyzed their composition by mass spectrometry. Our results indicate the presence of several  previously characterized ribosome biogenesis factors in complexes containing UTP6 and PNO1, as well as trypanosomatid-specific proteins that remain uncharacterized.  These findings provide insights into the composition of transient pre-ribosomal complexes from trypanosomatids, highlighting the participation of specific factors in earlier and later stages of biogenesis, some of which have not yet been elucidated. As a future objective, we aim to purify pre-ribosomal complexes through the application of additional chromatography steps, enabling their structural analysis by single-particle cryo-electron microscopy.