PRJNA875234
RUF6 ncRNA interacting proteins involved in P. falciparum immune evasion
Non-coding RNAs (ncRNAs) are emerging as important regulators of immune evasion and transmission of the human malaria parasite P. falciparum. RUF6 is a ncRNA gene family that is transcribed by RNA Polymerase III but regulates the activity of the Pol II transcribed virulence gene family called var. An understanding of how RUF6 ncRNA mechanistically connect to the downstream effectors is lacking. Here we developed a robust RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. Biotinylated antisense oligonucleotides were used to purify the RUF6 ncRNA interactome. Mass spectrometry identified several uniquely enriched proteins that are linked to gene transcription such as RNA Pol II subunits, nucleosome assembly proteins, and a homologue of the Dead-Box Helicase 5 (DDX5). Affinity purification of Pf-DDX5 identified several proteins originally found by our RUF6-ChIRP protocol, validating the robustness of the technique for the identification of ncRNA interactomes in P. falciparum. Inducible displacement of nuclear Pf-DDX5 resulted in the significant down-regulation of the active var gene. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression. Among the binding proteins, we identify a helicase that may resolve G-quadruplex secondary structures highly enriched in var genes to facilitate transcriptional activation and progression.