RNA Binding Proteins and Gene Expression Regulation in Trypanosoma cruzi

Abstract

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.

Keywords: CRISPR/CAS; RNA binding proteins; RNA granules; Trypanosoma cruzi; gene expression regulation; zinc finger protein.

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.

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 anti-TcAKR (1:1,000). TcAKR (Aldo-keto reductase, TCDM_00490) was used as a normalizer. A portion of the Ponceau-S stained blot is shown to demonstrate sample input. (D) 3xFLAG C-terminally tagged TcZC3H29 and TcZC3HTTP detection during metacyclogenesis. Protein extracts were prepared from 5 × 10⁶ parasites at different metacyclogenesis stages and incubated with anti-FLAG antibodies (1:1,000). A₂₄–nutritionally stressed adhered epimastigotes after 24 h; A₇₂–nutritionally stressed adhered epimastigotes after 72 h.

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.

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.

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 from genomic DNA, cloned into the pGEM-T Easy vector (Promega) and transformed into the TOP10 chemically competent Escherichia coli strain. Then, plasmids were isolated from the positive clones (identified by PCR and BglII digestion) and sent for sequencing for gene disruption confirmation. (D) Confirmation of single cell sorted clones containing the tczc3h39, tczc3h29, or tczc3http disrupted genes. Genomic DNA from the single cell cloned (by flow cytometry) population was used for tczc3h39, tczc3h29, or tczc3http PCR amplification followed by BglII digestion. At least one clone is representatively shown for each gene according to the specific gRNA used to achieve gene knockout (indicated above).

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.