RNA Binding Proteins and Gene Expression Regulation in Trypanosoma cruzi

Figures

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  Figure 1. General flow of RBPs action on the mRNA metabolism. Nuclear RBPs interact with their target mRNAs as soon as they start to be transcribed by RNA polymerase II (RNA pol II) and modulate mRNA maturation (trans-splicing and polyadenylation) and nuclear exportation processes. When the mRNAs arrive into the cytoplasm, cytoplasmatic RBPs (along with the ones that came from nucleus) engage their targets and (in a combinatorial signaling) determine the mRNAs fate by directing them to the translational machinery or to RNP granules for storage or degradation. The dynamic destination and trade of mRNAs between those fates relies according to the cellular condition. When submitted to stress (e.g., nutrients availability, pH, temperature), stress granules appear, and in order to recover cell homeostasis, RBPs play a major role in rearranging RNA granules composition by dynamically changing the mRNAs that will address to translation machinery, storage in stress granules or degraded in P-bodies. RBP, RNA binding protein (round shapes); NPC, nuclear pore complex; SG, stress granules; P-body, processing bodies.

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  Figure 2. The T. cruzi zinc finger proteins TcZC3H29 and TcZC3HTTP. (A) Graphic representation and amino acid sequences of the proteins. The zinc finger C3H domains are highlighted in green (with the cysteine and histidine residues underlined in bold) and the TcZC3HTTP DNAJ domain in blue. (B) Immunolocalization of TcZC3H29 and TcZC3HTTP. Epimastigotes were incubated with anti-TcZC3H29 (1:300) or anti-TcZC3HTTP (1:750), and an Alexa 488-conjugated goat anti-mouse antibody (1:600) was used for detection. The nucleus and kinetoplast are stained with DAPI. Bar = 10 μm. (C) TcZC3H29 and TcZC3HTTP expression profiles during metacyclogenesis. Western blot of protein extracts obtained from 5 × 106 parasites in distinct differentiation stages. Epimastigotes (E), epimastigotes after 2 h of nutritional stress (S), nutritionally stressed epimastigotes in the adhesion stage (A) and metacyclic trypomastigotes (M). To further investigate the expression of TcZC3H29 and TcZC3HTTP in the infective form, increased amounts of metacyclic trypomastigotes extract (5 × 106, 1 × 107, and 1.5 × 107 parasites) were used. Detection was performed with antisera against TcZC3H29 (1:500) and TcZC3HTTP (1:1,000) and…

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  Figure 3. Knockout of TcZC3H39, TcZC3H29, and TcZC3HTTP results in T. cruzi morphological changes. Parasites were visualized by light microscopy (upper lane) and scanning electron microscopy in higher or lower magnifications (middle and bottom lanes, respectively) 3 days after transfection with specific gRNAs targeting TcZC3H39 (A), TcZC3H29 (B), or TcZC3HTTP (C). Parasites expressing Cas9-GFP were also transfected with a non-guide RNA (RNA Control) as an experimental control (D). Arrow heads are indicating smaller flagella Bar: 10 μm.

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  Figure 4. Cell cycle impairment by disruption of the genes encoding TcZC3H39, TcZC3H29 and TcZC3HTTP. Populations transfected with specific gRNAs to (A) TcZC3H39, (B) TcZC3H29, or (C) TcZC3HTTP underwent cell cycle assessment by flow cytometry from day 1 to 5 post transfection (1–5 dpt). Graphs show the percentage of total cells (y-axis) by the amount of DNA (x-axis) in each transfected population line, with specific gRNAs, and on each day of analysis (rows). (D) A non-guide RNA (control RNA) was used as the CRISPR/Cas 9 system specificity control. Each graph also shows the wild-type population cell cycle (black dotted) for comparison.

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  Figure 5. Disruption of the genes encoding TcZC3H39, TcZC3H29, and TcZC3HTTP by CRISPR/Cas9 using a DNA donor strategy. (A) Scheme depicting the DNA donor strategy used for gene knockout. Zinc finger genes (represented on the left as TcZC3H39, TcZC3H29, and TcZC3HTTP) were targeted by Cas9 (through specific gRNA recognition) in different regions (the positions are indicated below each gene representation) along with a DNA template containing a 30-nt homology arm (at each end), a BglII restriction site (AGATCT) and a sequence that encodes stop codons in three different frames (TAGATAGATAG). (B) Genomic DNA from transfected populations with gRNAs targeting TcZC3H39, TcZC3H29, or TcZC3HTTP with (+) or without (–) the respective DNA donors was PCR amplified with specific primers for each zinc finger gene and digested with BglII for gene disruption visualization. Asterisks (*) highlight BglII restriction site incorporation by showing the digested products, whereas the remaining undigested amplicon is indicated by an arrow. (C) DNA sequencing of the tczc3h39, tczc3h29, and tczc3http genes showing correct insertion of the DNA donor. After transfection, zinc finger targeted genes were amplified…

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  Figure 6. TcZC3HTTP knockout confirmation after transfection with specific gRNAs. (A) 5% polyacrylamide gel electrophoresis to confirm DNA donor incorporation in the TcZC3HTTP gene. Genomic DNA from cloned population (by flow cytometry) was used for tczc3http PCR amplification followed by BglII digestion (left panel). Scheme depicting the expected increase in TcZ3HTTP gene size and BglII site and stop codon sequence insertion. (B) Western blot assay to confirm TcZC3HTTP gene disruption. TcZC3HTTP Hemi-knockout cloned population were transfected once again with gRNAs targeting TcZC3HTTP (gRNA 99 or gRNA 231) or a non-guide RNA control (Ctrl) and parasites were harvested and protein content extracted. Polyclonal antibodies against TcZC3HTTP (1:1000) were used for protein detection. The amount of protein extract applied corresponded to 1 × 107 parasites and TcAKR protein was used as an input control. This experiment was performed twice.