Downregulation of the essential Rrp44 ribonuclease causes extensive ultra-structure cell modifications in Trypanosoma brucei
Rrp44/Dis3 is an essential protein conserved in all eukaryotes that functions in the maturation of many different RNA precursors and RNA surveillance. The Trypanosoma brucei Rrp44/Dis3 homologue (TbRRP44) is required for maturation of pre-rRNA, spliced leader, and U3 snoRNA precursors. Its depletion leads to inhibition of cell proliferation and eventually to cell death. In this work, we showed that TbRRP44 depletion causes a massive expansion of acidic and lysosome-derived vacuoles, enlargement of cell and nuclei sizes without changes in DNA content, mitochondrial inactivation, and autophagy induction. Consistently, 3D reconstructions using cryo-soft X-ray tomography revealed extreme vacuolation of the cytoplasm and numerous cellular alterations, including an increase in size and number of calcium-containing vesicles and lipid droplets. These multiple defects indicate that a combination of alterations converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be a final stage leading to the cell death triggered by TbRRP44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by deficiency of this essential ribonuclease in T. brucei.