Depletion of TbRRM1 induces RNA Pol II transcription-elongation impairment and R-loops accumulation

LEVY GABRIELA V; TEKIEL VALERIA; BAÑUELOS CAROLINA P; SABORIT JUAN I; NÍTTOLO ANALÍA G; SANCHEZ DANIEL O.

Paraná

Congreso; LIV Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

TbRRM1 is an essential SR-related RNA binding protein from Trypanosoma brucei, the causative agent of sleeping sickness. Previous studies from our lab indicate that TbRRM1 depletion leads to both decreased RNA Pol II transcription-elongation rate and compacted chromatin in a particular polycistronic transcription unit. In the present work we showed, by chromatin and RNA immunoprecipitation assays, that TbRRM1 is both recruited to chromatin and to specific RNAs. Given this association, we further characterized TbRRM1 binding properties in the presence of RNAse A, RNAse H and Actinomycin D. Interestingly, TbRRM1 recruitment to chromatin increased under these treatments, thus suggesting that RNA and chromatin compete for TbRRM1 binding. In addition, we showed by RTqPCR and chromatin fractionation, that the abundance of transcripts belonging to genes downregulated after TbRRM1 depletion, increases in the chromatin-associated RNA fraction. Finally, as the RNAse H results suggested that TbRRM1 binds DNA-RNA hybrid, we studied whether TbRRM1 knockdown induces the formation of R-loops. To this end, we performed indirect immunofluorescence assays with the S9.6 antibody. TbRRM1 depleted cells showed a significant increase in the number of positive intranuclear dots, thus suggesting that TbRRM1 prevents R-loops accumulation.Altogether, our results suggest that RNA Pol II transcription-elongation impairment, induced by TbRRM1 depletion, might be a consequence of RNA Pol II slowing down due to R-loops accumulation. Our hypothesis is that TbRRM1 helps to displace the nascent mRNAs from the site of transcription, which prevents the formation of R-loops.