Mutant/mutation
The mutant expresses a C-terminal TurboID::GFP-tagged version of DOZI

Protein (function)
DOZI is an RNA helicase that is highly up-regulated in female gametocytes and shows high sequence homology to the DDX6 family of RNA helicases.  It contains domains involved in RNA-binding and RNA-unwinding activity.  In other eukaryotes the DDX6 family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the transport of mRNA to degradation centers in the cell (P-bodies). These helicases are found in organisms as diverse as yeast (e.g., Dhh1p) and humans (e.g., RCK/p54).

DOZI has a central role in the silencing and maintenance of steady-state levels of a population of gametocyte-specific transcripts. The loss of DOZI severely affects the capacity of the parasite to store and stabilize a discrete subset of mRNAs in the female gametocyte, resulting in a failure to synthesize specific proteins and to complete normal zygote development.

Female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the protein orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex (DCA) in female gametocytes that associates with many of the affected mRNAs

Phenotype
In order to determine mechanisms that may control translation across host-to-vector transmission, we assessed the composition of the DOZI/CITH/ALBA (DCA) translationally repressive protein complex before and after transmission. We and others have identified components of the DCA complex in P. yoelii and P. berghei via IP/MS in asexual blood stages, gametocytes, and oocyst sporozoites either with or without crosslinking. However, it was not clear how the DCA complex may respond to transmission cues that are predicted to cause the release of its bound and regulated mRNAs and thus relieve translational control. Therefore, we employed proximity proteomics by fusing a variant of E. coli biotin ligase and GFPmut2 to the C-terminus of DOZI and ALBA4 as experimental bait proteins, or as an unfused control to account for highly abundant proteins in the same compartment (i.e., cytosol). We chose this approach to not only determine the protein composition of the DCA complex across host-to-vector transmission, but also to determine if spatial organization of this complex changes. We selected DOZI and ALBA4 as they are known to associate with the 5’ or 3’ end of bound mRNAs, respectively, and thus provide protein proxies for the two ends of target mRNAs.
We initially tested the first generation BioID enzyme before adopting the use of the further engineered TurboID (TID) ligase that allowed for more robust labeling in a shorter labeling time, as we and others have recently shown for P. falciparum and P. berghei.To establish an appropriate duration of labeling, we assessed in vivo biotinylation with supplementation with 150 μM biotin for increasing time, then used total parasite lysate for affinity blots probed with streptavidin-HRP for both bait parasite lines, ALBA4::TurboID::GFP and DOZI::TurboID::GFP. We found that one hour of biotin supplementation was sufficient for robust biotin labeling by TurboID in total parasite lysates for both female gametocytes and zygotes. Therefore, we used these labeling conditions for all experiments.
Biotinylated proteins were able to be captured from female gametocyte or zygote lysates on magnetic streptavidin beads for on-bead tryptic digestion and mass spectrometry analysis.

Evidence is presented that: 'the compositional and spatial rearrangements detected by proximity proteomics are consistent with DOZI/CITH/ALBA mRNPs 1) adopting a condensed state during translational repression in female gametocytes, and then 2) undergoing composition changes that promote its elongation in zygotes following the release from translational repression'.

Additional information

Other mutants