Downregulation of the essential Rrp44 ribonuclease causes extensive ultra-structure cell modifications in Trypanosoma brucei

Rrp44 is a conserved eukaryotic protein showing endonuclease and exoribonuclease activity that plays essential functions in RNA maturation and degradation. In , depletion of Rrp44 (TbRrp44) blocks processing of large subunit ribosomal RNA precursors, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We used a combination of molecular and chemical markers to investigate the fate of cells upon knockdown of TbRrp44. In addition, synchrotron deep UV microscopy and cryo soft X-ray tomography were used to investigate cell morphology and ultra-structure modifications. Downregulation of TbRrp44 results in induction of autophagy, inactivation of mitochondria and expansion of acidic and lysosome-derived vacuoles in parallel with enlargement of cell size. Nuclei also increase in size without changes in DNA content. 3D tomographic reconstructions revealed extreme vacuolation of the cytoplasm of TbRrp44 knockdown cells and general alteration of organelles. Calcium-containing vesicles (acidocalcisomes) were identified by X-ray absorption near-edge structure spectra (XANES) of calcium L edges on fully hydrated cells. The volumes of acidocalcisomes and lipid droplet were quantified from 3D reconstructions. Both were found in higher number and with larger volumes in TbRrp44 knockdown cells. These multiple defects indicate that a combination of signals, starting from nucleolar stress and activation of autophagy, converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be one of the final stages leading to cell death triggered by TbRrp44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by Rrp44 ribonuclease deficiency.