Authors
AT Menezes1; AM Silber1; JF Nascimento2; PP Coltri1; 1 Biomedical Science Institute - USP, Brazil; 2 Instituto de Ciências Biomédicas, BrazilDiscussion
Trypanosoma cruzi currently infects 6 to 7 million people worldwide, causing Chagas disease, which, when symptomatic, is characterized by cardiomyopathy, digestive system failure, or both. Trypanosomatids’ genes are transcribed as polycistronic pre-mRNAs, which are individualized by SL trans-splicing, by the addition of a spliced leader sequence (SL-RNA) to each ORF in these pre-mRNAs. SL trans-splicing is carried out by the spliceosome, which is composed of a core of 5 ribonucleoproteins (RNPs) and several regulatory proteins conserved among trypanosomes and other eukaryotes. However, details on the function and activity of specific proteins in trypanosomatids' spliceosomes remain largely unexplored. Previously, we analyzed the T. cruzi spliceosome by mass spectrometry. The present work intends to characterize two of the detected proteins, TcRRM (TcCLB.510143.80) and TcHEL67 (TcCLB.506213.120), which are orthologous to the human SF3b4 and DDX3X proteins, respectively. In humans, these proteins participate in several cellular processes, including pre-mRNA splicing regulation. We overexpressed the TcRRM and TcHEL67 proteins in T. cruzi epimastigotes, and used CRISPR-Cas9 to edit their coding sequences. We analyzed the variations in splicing efficiency of endogenous α-tubulin, GAPDH and Poly-A polymerase transcripts by RT-qPCR. Using immunofluorescence, we confirmed that the TcRRM and TcHEL67 proteins localize within the parasite's nucleus. Besides, immunoprecipitation assays showed that TcRRM seems to be associated to U2, U5 and U6 spliceosome snRNAs, while TcHEL67 do not show any strong association. Our findings suggest that the TcRRM and TcHEL67 proteins may have a role in regulating splicing in T. cruzi epimastigotes. We intend to further explore the results by in vitro splicing assays, and use CLIP-seq to check for RNA association for these proteins.
