Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

Abstract

BackgroundFollowing fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown.ResultsWe identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development.ConclusionsOur study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0493-0) contains supplementary material, which is available to authorized users.

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

“…2B and SI Appendix, Fig. S4), a behavior consistent with translational regulation during the transmission of P. berghei found for many genes (20)(21)(22). Storage of this mRNA in cytoplasmic mRNPs for translation in the ookinete was confirmed in an RNA immunoprecipitation (IP) experiment, in which dhhc10 was copurified with the translational repressors DOZI (a homolog of yeast DHH1p) and CITH (a homolog of yeast Scd6p) (Fig.…”

“…Following a mosquito blood meal, mating of males and females produces the round zygote that develops into a motile, banana-shaped ookinete. This transformation process relies heavily on translationally repressed (stored) mRNAs provided by the female gametocyte (20)(21)(22). Of 11 annotated P. berghei DHHC-PATs (17, 23) we have identified dhhc10, along with dhhc2 and dhhc3, as being up-regulated in gametocytes (Fig.…”

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.…”

“…Invitrogen protocol and used in PCR in order to detect transcription of GFP-tagged alleles and known gametocyte transcripts [25]. Primer pairs were as follows: …”

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 .…”