Molecular Targeted knockdown of the RNA-binding protein TbRRM1 from Trypanosoma brucei induces cell-cycle arrest in G1/S phase and programmed cell death.

MENDIONDO N; ORTIZ GE; MORETTI G; DOCAMPO R; SÁNCHEZ DO

Marine Biology Laboratory, Woods Hole, MA, United States

Congreso; (23rd) Annual Molecular Parasitology Meeting; 2012

TbRRM1 is an essential RNA binding protein from Trypanosoma brucei belonging to the SR-related protein family. SR proteins have been extensively studied in eukaryotes given their relevance in key cellular processes like constitutive and alternative splicing of pre-mRNA and processes beyond splicing such as nonsense-mediated mRNA decay and translation. In the present study, we show that TbRRM1 depletion by RNAi in the procyclic form arrest the growth after 24hs of induction. A Concomitant appearance of elongated cells was also detected in this population. Subsequent examination of the number of nucleus and kinetoplasts per cell, toghether with FACS analyses enabled us to determine that the posterior-end elongated cells were arrested in G1/S cell-cycle phase, showing nozzle phenotype. This phenotype was also test with the YL 1/2 antibody that recognizes tyrosinated tubulin present in newly assembled microtubules. Interestingly, following these initial events we confirmed that most TbRRM1 RNAi-induced cells underwent programmed death. Our results suggest that deficiency of TbRRM1 affects cell cycle progression, which, in turn, leads to programmed cell death. Given the general characteristics of the TbRRM1 protein, like its structure and nuclear spleckled-like distribution, it was previously suggested that TbRRM1 may play a role in mRNA metabolism. For this reason, we decided to search for regulation of genes possibly involved in the phenotypes observed TbRRM1-depleted cells by microarray. The comparison of the mRNA levels of TbRRM1-depleted cells with that of uninduced cells show that most of the mRNAs analyzed decreased after RNAi induction but surprisingly others increase.