TbRRM1 have opposite roles in transcriptional regulation of RNA Pol II-transcribed units in Trypanosoma brucei.

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

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

SAIB-SAP-SAIC-SAi, etc

TbRRM1 HAVE OPPOSITE ROLES IN TRANSCRIPTIONAL REGULATION OF RNA POL II-TRANSCRIBED UNITS IN Trypanosoma brucei 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 ablation by RNAi in procyclic cells leads to cell-cycle block and cell death by an apoptotic-like mechanism. Recently, TbRRM1 was found associated to numerous mRNAs and to core histones, which suggests a dual role in both transcriptional and post-transcriptional regulation. To determine if TbRRM1 is involved in the trans-splicing process, we first evaluated the steady-state level of the SL-RNA by RTqPCR in parasites depleted or not of TbRRM1. The SL-RNA level increased after RNAi induction relative to uninduced parasites, suggesting either a global trans-splicing defect or an up-regulation of the SL-RNA by increased transcription. To evaluate the trans-splicing efficiency in parasites depleted of TbRRM1, we then determined by RTqPCR, the pre/mature mRNA ratio of TbNOP86 and 60S ribosomal protein L38, known to be down-regulated after TbRRM1 knockdown. Both pre and mature mRNA levels were reduced after TbRRM1 silencing indicating a transcriptional impairment induced by TbRRM1 knockdown, rather than a trans-splicing defect, which was not affected. We then carried out Run-On assays to evaluate if TbRRM1 is involved in gene transcription regulation. We found a global decrease of RNA synthesis after RNAi induction. Moreover, transcription analyses of neighboring RNA Pol II-dependent protein-coding genes showed that TbRRM1 is required for transcription elongation. Interestingly, our results also showed that transcription of the SL-RNA was significantly increased in parasites depleted of TbRRM1, which correlated with increased SL-RNA levels.In summary, our data show that TbRRM1 could be involved in both facilitating transcription elongation of polycistronic transcription units and repressing expression of monocistronic units of the SL-RNA genes, both process mediated by RNA Pol II. Keywords: RNA binding protein, trans- splicing, transcription elongation, epigenetic regulation, chromatin remodeling.