TbRRM1 silencing leads to DNA synthesis impairment and disturbs normal cell cycle progression in procyclic Trypanosoma brucei.

LEVY, GABRIELA VANESA; BAÑUELOS, CAROLINA; NÍTTOLO, ANALÍA; TEKIEL, VALERIA; SÁNCHEZ, DANIEL

Mar del Plata

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias.; 2014

TbRRM1 SILENCING LEADS TO DNA SYNTHESIS IMPAIRMENT AND DISTURBS NORMAL CELL CYCLE PROGRESSION IN PROCYCLIC Trypanosoma brucei Gabriela V. Levy, Carolina P. Bañuelos, Analía G. Níttolo, Valeria S. Tekiel and Daniel O. Sánchez. TbRRM1 is an essential RNA binding protein from T. brucei which belongs to the SR-related protein family. Previous studies from our lab indicated that TbRRM1 RNAi knockdown in procyclic cells disturbed normal cell cycle progression by arresting cells at G1 phase 24h post-RNAi induction, with the concomitant emergence of nozzled parasites which present an abnormal posterior cell end elongation. Here, we provide evidence that after TbRRM1 silencing DNA synthesis was severely compromised since BrdU incorporation dropped ~50% after 2 days of RNAi induction. Analysis of cell viability, using Rhodamine 123 and propidium iodide, showed that the number of viable cells decreased significantly only after 72h of RNAi induction, indicating that early DNA synthesis impairment is a primary effect caused by TbRRM1 ablation and not a consequence of parasite death. Nucleus-kinetoplast (NK) configuration studies showed that after TbRRM1 depletion, parasites with misplaced 2N2K arose along with 2N1K and 0N1K aberrant populations, indicating a disturbed mitotic or post-mitotic phase. Moreover, the nozzled parasite group presented an enrichment of the one nucleus-one elongated kinetoplast (1N1K*) configuration suggesting that kinetoplast segregation might be also affected. As shown previously, Annexin-V and cell-cycle analysis results indicated that TbRRM1 silencing led to programmed cell death. Here, we show that after TbRRM1 RNAi induction the cell population with reduced mitochondrial membrane potential increased significantly, supporting the notion that cell death is mediated through an apoptotic mechanism. Altogether, our results indicate that depletion of TbRRM1 leads to DNA synthesis impairment and both cell-cycle arrest at G1 and disturbance of the mitotic/post-mitotic phase suggesting that TbRRM1 is involved in cell cycle control and progression, although its direct or indirect connection to the cell cycle regulation remains to be elucidated.