Identification of novel partners of the SR-related protein TbRRM1 in Trypanosoma brucei.

ROJAS, J; NITTOLO, A; BAÑUELOS, C; SABORIT, J; TEKIEL, V; SÁNCHEZ, DO; LEVY, GV

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias.; 2017

SAIB-SAP-SAIC-SAi, etc

IDENTIFICATION OF NOVEL PARTNERS OF THE SR-RELATED PROTEIN TbRRM1 IN Trypanosoma bruceiPreviously, we have demonstrated that TbRRM1 silencing leads to cell cycle block, apoptotic-like death and abnormal cell elongation in the procyclic stage of T. brucei.TbRRM1 has three RNA binding motif, two zinc fingers and one small C-terminal SR domain potentially involved in protein-protein interaction. Recently, TbRRM1 has been shown to retain numerous RNAs in nucleus avoiding their translation. In addition, TbRRM1 was also associated with a few protein partners including core histones and several hot spot proteins. However, these results were obtained from an in-gel digestion assay, leading to a partial identification of the associated proteins. Therefore, to obtain a more complete view of this interaction map we addressed the identification of TbRRM1 partners by liquid chromatography tandem-mass spectrometry approach (LC-MS/MS). For that purpose, we carried out the N-terminal tagging of one allele of the TbRRM1 gene in RNAi-TbRRM1 procyclic cells. It is worth mentioning that parasites conditionally express the RNAi which corresponds to the 5´UTR fragment of the TbRRM1 gene present only in the wild type allele, therefore allowing the unaffected expression of the tagged version. The tagged protein rescued the lethal phenotype caused by the depletion of the TbRRM1 protein, since no differences were observed in the growth curve of the induced parasites relative to the uninduced cultures. Moreover, immunofluorescence analysis with anti-flag monoclonal antibody and anti-TbRRM1 serum evidenced the correct localization of the tagged protein in parasites from TET+ cultures. Altogether, these results indicate that the tagging does not interfere with either protein localization or TbRRM1 function. Pull down of TbRRM1 was carried out by anti-flag antibody and samples were subjected to LC-MS/MS. Partners identified in this work, will contribute to elucidate the function of TbRRM1 protein to gain a better understanding about Trypanosoma biology.Keywords: RNA BINDING PROTEIN, PROTEIN INTERACTION, GENE EXPRESSION REGULATION.