Regulation of Sexual Development of Plasmodium by Translational Repression

Abstract

Translational repression (TR) of mRNAs plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. We show here that TR and mRNA turnover are key influences on stage specific gene expression in the protozoan Plasmodium. The DDX6 class RNA helicase, DOZI (development of zygote inhibited), is found in cytoplasmic bodies of female, blood stage gametocytes in a complex with mRNA species known to be translationally repressed. Genetic disruption of pbdozi inhibits the formation of translationally quiescent mRNPs and targets these and other (>350 different) transcripts to the degradation pathway rather than directing them to translating polysomes, preventing zygote development prior to meiosis in the mosquito. Thus TR is essential in malaria parasite development. A full catalogue of the proteins and processes associated with DOZI (the first described malaria parasite TR-effector protein) might lead to novel approaches to prevent parasite development.Translational repression (TR) of mRNAs in higher eukaryotes controls temporal expression of specific protein cascades or directs the location of translation within a cell, and is prominent after gamete fertilisation (zygote formation) in the early embryo when de novo transcription of mRNA is restricted (1-5). The hallmark of repression is the assembly of certain mRNAs together with proteins into quiescent messenger ribonucleoprotein particles (mRNPs) where these transcripts are stored for translation at a later time. 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 * To whom correspondence should be addressed waters@lumc.nl. Post-fertilsation development of Plasmodium zygotes in the mosquito relys on the formation of translationally quiescent mRNPs in blood-stage, female sexual precursor cells Supporting Online Material www.sceincemag.org Materials and Methods Figs. S1 to S4 Tables S1 to S4 References

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

“…Translational repression mediated by DOZI and CITH is essential for P. berghei ookinete development [17,18,25] and we believe that similar mechanisms play a role in the human parasite. In line with this hypothesis we have broadly defined a regulatory function for the 3" flanking regions of two P. falciparum transcripts: pfs28 and the cpw-wpc gene PF3D7_1331400 in gametocytes wherein translation is upregulated upon activation of gametocytes.…”

“…Transcripts of the genes p25, p28 as well as ap2-o, a key transcription factor during ookinete maturation [42], are assembled into cytoplasmic mRNPs that can be visualized by FISH [18] and are co-immunoprecipitated with the repressors DOZI and CITH [17]. The DEAD-box RNA helicase DOZI (Development of Zygote Inhibited) [18] and the LSm protein CITH (CAR-I and Trailer Hitch homolog) [17] are core components of cytoplasmic messenger ribonucleoprotein particles (mRNPs) that function in the storage and stabilization of known repressed mRNAs in the female gametocyte, but are ready for rapid translation during ookinete formation after transmission of the parasite to the mosquito vector [17,18,[38][39][40]43].…”

“…The DEAD-box RNA helicase DOZI (Development of Zygote Inhibited) [18] and the LSm protein CITH (CAR-I and Trailer Hitch homolog) [17] are core components of cytoplasmic messenger ribonucleoprotein particles (mRNPs) that function in the storage and stabilization of known repressed mRNAs in the female gametocyte, but are ready for rapid translation during ookinete formation after transmission of the parasite to the mosquito vector [17,18,[38][39][40]43]. Several P. berghei cpw-wpc transcripts were present in the pool of genes affected in cells depleted of DOZI and CITH (Table 4) [17,18]. To test whether cpw-wpc mRNAs of P. berghei are bound in DOZI/CITH-defined mRNPs we used transgenic parasite lines that express GFP-tagged DOZI or CITH [17] in RNA immunoprecipitation (RIP) experiments (Figure 2).…”

“…Immunoprecipitation (IP) using P. berghei DOZI::GFP and CITH::GFP gametocyte extracts and subsequent RNA extraction, reverse transcription and PCR of IP fractions were carried out as described [17,18]. Primers used in RT-PCR assays are shown in Table 1.…”

The development of malaria parasites in the mosquito midgut

“…In P . berghei , a total of 731 mRNAs associate with the RNA helicase DOZI (development of zygote inhibited) and the Sm‐like factor CITH (homolog of worm CAR‐I and fly Trailer Hitch), which function in translational transcript repression, including mRNAs encoding for the GPI‐anchored female‐specific protein P25 and the glideosome‐associated proteins GAP45 and 50 (Mair et al ., 2006, 2010; Guerreiro et al ., 2014). For P .…”