Characterization of the RNA-Binding Protein TcSgn1 in Trypanosoma cruzi

Figure 1. TcSgn1 protein secondary structure. Structural prediction was performed against Saccharomyces cerevisiae Sgn1 protein. Confidence was 99.98%, and identity was 45%. (top) Predicted structure, and colors refer to alignment confidence. (bottom) Amino acid sequence alignment; RRM domain indicated by black line, RRM core indicated in a black box, and RGG region indicated in dark orange. Confidence values obtained from probabilities calculated by Phyre2 software.

Figure 2. TcSgn1 is developmentally regulated throughout the T. cruzi life cycle. (A) Western blot of Trypanosoma cruzi throughout metacyclogenesis. Primary antibodies used: Sgn1:FLAG-N:anti-FLAG M2 (Sigma–Aldrich) 1:1000 dilution and anti-DHH1 (used as protein normalizer) 1:500. Secondary antibody: antimouse AP (Sigma–Aldrich). Epi: epimastigotes in the exponential phase of growth, Str: epimastigotes under nutritional stress, Ad. 24 h: epimastigotes adhered for 24 h, Met: metacyclic trypomastigotes. (B) Protein-relative abundance for quantitative proteomics data plotted in log2 fold change (y axis). Ratios represented in x axis; Str/Epi—stressed epimastigotes/epimastigotes; Ad/Epi—adhered epimastigotes/epimastigotes; Meta/Epi—metacyclic trypomastigotes/epimastigotes. TcDhh1 used as control for Western blot and quantitative proteomics. (C) Expression levels of Sgn1 mRNA in epimastigote and trypomastigote forms obtained from RNA-seq data and compared total RNA with ribosome profile. Transcript level expressed in fragments per kilobase per million (FPKM). (D) Cellular localization of TcSgn1. Primary antibody: anti-FLAG M2 (Sigma–Aldrich) 1:500. Secondary antibody was Alexa Fluor-conjugated…

Figure 3. Cellular localization of TcSgn1. Primary antibody: anti-FLAG M2 (Sigma–Aldrich) 1:500. Secondary antibody was Alexa Fluor-conjugated antimouse 488 (green, 1:400; Thermo Fisher). Arrows indicate granules in cytoplasm. Kinetoplasts and nuclei labeled with DAPI in blue (4′,6-diamidino-2-phenylindole, dihydrochloride) at a 1:1000 ratio. Epi: epimastigotes in exponential phase of growth, Str: epimastigotes under nutritional stress and sinefungin treatment for 30 min.

Figure 4. Heat map representing normalized expression levels (transcripts per million) of 25 most enriched mRNAs associated with TcSgn1 in epimastigotes (false discovery rate (FDR) < 0.1% and fold change > 10-fold). Each row represents the biological replicates for each condition. Expression levels visualized using gradient color scheme: blue—high expression level; red—low expression level.

Figure 5. RNA-binding assay in TcSgn1 from T. cruzi. (A) Pulldown with oligo-dT beads. Input = total cell extract; FT = supernatant after incubation with poly(T) beads, eluate = eluted. Assay was performed with (+) and without (−) competition using 10 mg/mL poly(A). (B) RNA-binding assay in vitro. TcSgn1 concentration varied from 500 ng to 3 µg of soluble recombinant protein. A poly(A) unlabeled probe was used as a competitor and a control of nonspecific binding.

Figure 6. Map of proteins associated with TcSgn1 and in vitro binding validation assay. (A) TcSgn1 interaction map obtained from String database. (B) Coimmunoprecipitation of PABP1 and TcSgn1 in T. cruzi TcSGN1-flag strain (epimastigotes) and control of coimmunoprecipitation of GFP-flag strain and PABP1 or TcSgn1 in transfectant epimastigotes. (C) Antibody specificity control: WT—wild-type epimastigote extract. Primary anti-PABP1 antibody (1:500) and primary anti-FLAG M2 antibody (Sigma-Aldrich) (1:1000) were used. Secondary antirabbit peroxidase antibody (1:10,000) revealed in L-PIX CHEMI EXPRESS (Loccus) device.