Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development

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.

Figure 1. Identification of a multiprotein complex engaged in storage of translationally silent mRNAs in female Plasmodium gametocytes.

A and C Western blot analysis of DOZI and CITH immunoprecipitation (IP) eluates show specific isolation of the respective GFP fusion proteins. P47, a female gametocyte specific protein, did not co-IP and is present only in input fractions. Equivalent amounts were loaded. B Silver staining of the DOZI::GFP IP eluate separated on a 12% SDS-PAGE. The asterisk indicates the position/size of the DOZI::GFP fusion protein. D Reciprocal IP targeting C-terminal GFP-fusion proteins of DOZI and CITH resulted in the pull down of a set of proteins that were identified using LC-MS/MS. Shown are all proteins with conserved motifs (drawn to scale) and – grey underlaid – the number of unique peptide hits/protein in specific anti-GFP and control IPs. Alignments of these factors and additional Plasmodium-specific factors are shown in Table S1, Figures S1, S4, S5, S6, S7, S8, S9 and S10. Homology was defined on amino acid level, as well as domain presence and architecture.

Figure 2. Phylogenetic position of Plasmodium Alba-domain proteins.

We generated a multiple sequence alignment of the conserved region of a range of Alba-domain proteins (PFAM PF01918). The phylogenetic tree was generated with PHYML. Bootstrap values (100 replicates) are based on neighbor joining and maximum likelihood analyses. Accession numbers are from uniprot; species abbreviations are for Perkinsus marinus (9ALVE), Cryptosporidium muris (9CRYT), Trypanosoma brucei (9TRYP), Aeropyrum pernix (AERPE), Archaeoglobus fulgidus (ARCFU), Babesia bovis (BABBO), Dictyostelium discoideum (DICDI), Plasmodium berghei (PLABE), Plasmodium falciparum (PLAF7), Stylonychia lemnae (STYLE), Sulfolobus solfataricus (SULSO), Theileria parva (THEPA) and Toxoplasma gondii (TOXGO).

Figure 3. CITH co-localizes with translationally repressed mRNAs.

A Northern analysis of CITH::GFP IP eluates show specific co-elution of translationally repressed mRNAs p25 and p28, but not transcripts known to be translated (p47, eef1a) or ribosomal RNA. Equivalent amounts of eluates and input were loaded. B RT-PCR analyses show specific enrichment of transcripts known to be translationally repressed in the anti-GFP pull down; transcripts of expressed proteins (P47, DOZI, CITH) are absent. C The CITH::GFP fusion protein is present in characteristic foci in the cytoplasm of live parasites. D Fluorescent in situ hybridization combined with immunofluorescence analysis shows overlapping signals for p25 and p28 mRNAs and CITH::GFP. Scale bar  = 4 µm.

Figure 4. Zygotes formed by Δpbcith and Δpbdozi gene deletion mutants abort zygote to ookinete transformation.

A Schematic representation of the vector used to introduce the gfp/rfp male/female expression cassette into the p230p locus. B Identification of populations of RFP+ female gametocytes and GFP+ males by FACS in blood infected with parasites of line 820cl1m1cl1. Gametocyte populations are clearly separated from the population of red blood cells and red blood cells infected with the asexual bloods stages (asterisk). The inset shows male (GFP+) and female gametocytes (RFP+). Scale bar  = 4 µm. C RFP and Hoechst 33258 staining of female gametocyte, zygote and ookinete of line 820cl1m1cl. Zygotes and ookinetes were collected at 4 hours post activation (hpa) and 18 hpa, respectively. Note the increased Hoechst fluorescence intensity of the nuclei of zygotes and ookinetes as a result of fertilization and meiotic DNA replication, resulting in tetraploid nuclei. Scale bar  = 2 µm. D and E FACS analysis of Hoechst fluorescence intensity of wild type female gametes (D) and zygotes (E). In (D) haploid female gametes are shown before fertilization and (E) shows unfertilized females (1N) and tetraploid zygotes (4N) collected at 4 hpa. F Δpbdozi females are fertilized as shown by the doubling in DNA content, but do not achieve tetraploidy. G Females of Δpbcith mutants develop into tetraploid forms.

Figure 5. CITH and DOZI gene deletion mutant gametocytes suffer substantial mRNA loss.

A Northern blot analysis of four translationally repressed transcripts in wild type and Δpbcith mutants shows a clear de-stabilizing effect in the absence of CITH in gametocytes. B Venn diagrams of all 2-fold differentially expressed genes common to DOZI and CITH KO parasites. Hierarchical cluster analysis of the differentially expressed genes common to both lists. C Absence of DOZI or CITH does not result in the precocious translation of p28 mRNA into protein in female gametocytes.