Discovery of RUF6 ncRNA–interacting proteins involved in<i>P. falciparum</i>immune evasion

Abstract

Non-coding RNAs (ncRNAs) are emerging regulators of immune evasion and transmission of Plasmodium falciparum . RUF6 is an ncRNA gene family that is transcribed by RNA polymerase III but actively regulates the Pol II–transcribed var virulence gene family. Understanding how RUF6 ncRNA connects to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA–protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including RNA Pol II subunits, nucleosome assembly proteins, and a homologue of DEAD box helicase 5 (DDX5). Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum . Inducible displacement of nuclear Pf-DDX5 resulted in significant down-regulation of the active var gene. Our work identifies a RUF6 ncRNA–protein complex that interacts with RNA Pol II to sustain the var gene expression, including a helicase that may resolve G-quadruplex secondary structures in var genes to facilitate transcriptional activation and progression.