A seven-helix protein constitutes stress granules crucial for regulating translation during human-to-mosquito transmission of Plasmodium falciparum
Fig 7
Complex formation of 7-Helix-1 with Puf2 and repressed pfs25 mRNA and Pfs25 synthesis.
(A) Co-immunoprecipitation of 7-Helix-1 with Puf2. Co-immunoprecipitation assays on lysates of 7-Helix-1-HA or WT NF54 gametocytes were employed using either polyclonal mouse anti-Puf2 or anti-Pf39 antisera, followed by WB using rabbit anti-HA antibodies or mouse anti-Pf39 antibody to detect precipitated proteins. Black arrows indicate precipitated proteins, grey arrows indicate expected running lines in the negative controls. Asterisks indicate bands corresponding to the precipitation antibodies. Results are representative of three independent experiments. (B) Association of repressed transcript with 7-Helix-1. RIP assays on lysates of 7-Helix-1-HA gametocytes were employed using either rabbit anti-CITH or rabbit anti-HA antibodies, followed by RNA isolation. RT-PCR was performed amplifying co-eluted transcripts of ccp2 (198 bp), pfs25 (459 bp) and pfs28 (494 bp). cDNA preparation lacking the reverse transcriptase (-RT) was used to prove absence of gDNA contamination. (C, D) Quantitative expression analysis of gametocyte-specific adhesion proteins. (C) Expression of Pfs25 and Pfs230 in activated gametocytes. WT NF54 and 7-Helix-1-KO 2E6 gametocytes at 30 min p.a. were immunolabeled with rabbit anti-Pfs25 (green) and mouse anti-Pfs230 antibodies (red). Nuclei were highlighted by Hoechst33342 nuclear stain (blue). Bar, 5 μm. (D) Quantitative IFA analysis of Pfs25 and Pfs230. IFAs were employed as described above and the fluorescence intensities for Pfs25 and Pfs230 of 20 immunolabeled activated gametocytes were measured in triplicate using ImageJ (mean ± SD). The respective signal in WT lysates was set to 1. *** p ≤ 0.001 (Student‘s t-test). Results (in A-C) are representative of two to three independent experiments.