Abstract
A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.
Highlights
Post-fertilization development in multi-cellular organisms relies on mRNAs supplied in the oocyte in translationally silent P body related storage particles known as P granules
Through immunoprecipitation (IP) of DOZI::GFP followed by RNA analysis of IP eluates by Northern and RT-PCR analysis we have previously shown a clear physical association of this DEAD-box RNA helicase with mRNAs known to be translationally silenced in mature, female gametocytes [9]
In two independent IP experiments targeting DOZI::GFP a complex from Plasmodium berghei gametocytes was purified and analyzed by LCMS/MS yielding a group of DOZI interaction partners (Figure 1AB,D; Table S1); one of the co-eluted proteins, PB000768.03.0, showed strong homology with worm CAR-I and fly Trailer Hitch and Xenopus Rap55; these proteins co-localize with their respective DOZI homologues CGH-1 and Me31b to germ cell and P granules [3,4,5,6,7] – the Plasmodium protein contains both the conserved LSM14 domain and the extended FDF motif (Figures 1D, S1) known to compete with the enhancer of mRNA decapping EDC3 for binding to DDX6 helicases [15], and is designated CITH (CAR-I/Trailer Hitch Homolog; Figure S1)
Summary
Post-fertilization development in multi-cellular organisms relies on mRNAs supplied in the oocyte in translationally silent P body related storage particles known as P granules. Translation of these maternal mRNA pools depends on fertilization and occurs prior to maternal to zygote transition when transcription from the zygotic genome is initiated [1,2]. Plasmodium are haploid throughout most of their life cycle and sexual development in malaria parasites is initiated with the generation of sexual precursor cells, or gametocytes, in the blood of the mammalian host These mature, haploid male or female forms present distinct proteomic profiles [8] in the absence of sex chromosomes. In the mosquito midgut fertilization yields a diploid zygote that undergoes meiosis without cell division resulting in a tetraploid cell that within 18 hours transforms into the motile
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