Dataset Information

Identification of Plasmodium yoelii ALBA4 Binding partners by Immunoprecipitation/nano LC-MS/MS

ABSTRACT: RNA-binding proteins, such as DOZI/CITH and Puf2, have been shown to play critical roles in the life cycle in Plasmodium species. One of the characterized functions of these RNA-binding proteins is to bind to mRNAs and regulate their fates, and it is hypothesized that this regulation of mRNA homeostasis of specific transcripts is important for successful infection of both vectors and hosts by these parasites. To further understand the role that other RNA-binding proteins may play in translational repression and other critical processes in the parasite, we investigated ALBA4, another RNA-binding protein implicated in translational repression. We appended a C-terminal GFP-tag to ALBA4 (ALBA4::GFP), and then performed immunoprecipitations on chemically cross-linked samples using Streptavidin-coated beads coated with a biotin-conjugated anti-GFP antibody to capture the ALBA4 complex(es). We performed these experiments in multiple life cycle stages, including sexual stages (gametocytes) and asexual stage (schizonts). In gametocytes, we determined that ALBA4 associates with translational repression machinery. This is also the case in schizonts, however ALBA4 also associates with complexes involved in active translation, mRNA export, and mRNA degradation. We hypothesize that ALBA4 plays a multi-faceted role in mRNA homeostasis by associating with multiple protein complexes in a stage-specific manner.

OTHER RELATED OMICS DATASETS IN: PRJNA322665

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Plasmodium Yoelii

SUBMITTER: Scott Lindner

LAB HEAD: Scott Eugene Lindner

PROVIDER: PXD004183 | Pride | 2017-08-28

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	151027ASLSAM01407_SEL15.raw	Raw
	151027ASLSAM01408_SEL16.raw	Raw
	151201ASLSAM01460_SEL21.raw	Raw
	151201ASLSAM01461_SEL22.raw	Raw
	151201ASLSAM01462_SEL23.raw	Raw

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Publications

ALBA4 modulates its stage-specific interactions and specific mRNA fates during Plasmodium yoelii growth and transmission.

Muñoz Elyse E EE Hart Kevin J KJ Walker Michael P MP Kennedy Mark F MF Shipley Mackenzie M MM Lindner Scott E SE

Molecular microbiology 20170829 2

Transmission of the malaria parasite occurs in an unpredictable moment, when a mosquito takes a blood meal. Plasmodium has therefore evolved strategies to prepare for transmission, including translationally repressing and protecting mRNAs needed to establish the infection. However, mechanisms underlying these critical controls are not well understood, including whether Plasmodium changes its translationally repressive complexes and mRNA targets in different stages. Efforts to understand this hav ...[more]

PMID: 28787542

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Project description:The transmission of the malaria parasite between mosquitoes and mammals requires translational repression to ensure that only the proper proteins are expressed at the right time, while still allowing the parasite to prepare the mRNAs it will need for the next developmental stage. With relatively few known specific transcription factors (ApiAP2 family) that may specifically initiate gene transcription, Plasmodium parasites also regulate the stability and turnover of transcripts to provide more comprehensive gene regulation. The CAF1/CCR4/NOT complex has been shown in model organisms to be important for not only mRNA degradation, but also translational control through its deadenylases CAF1 and CCR4. However, few proteins that impose translational repression in Plasmodium sexual stages are known, and those that are characterized primarily affect female gametocytes. Therefore, we have characterized two deadenylases and have uncovered their roles in transmission. We have identified and characterized CCR4-1, which we show plays a role in activating male gametocytes, stabilizing transcripts in gametocytes, and regulating host-to-vector transmission. We find that when ccr4-1 is genetically deleted, there is a loss in the coordination of male gametocyte activation and a reduction in the ability of the parasite to productively infect the mosquito, which is independent of its effect upon male activation. Comparative RNA-seq shows that the deletion of ccr4-1 affects many transcripts that are translationally repressed in females, are related to male gamete function, and/or are important for early mosquito stage development. In contrast, we found that genetic deletion of the major deadenylase Caf1 is lethal. However, we observed that expression of only the N-terminal Caf1 domain is permissive, yet prevents proper complex assembly and phenocopies the ccr4-1 deletion. We therefore conclude that the general and transmission-specialized deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in parasite growth and transmission.

Project description:For malaria transmission, the parasite must undergo sexual differentiation into mature gametocytes. However, the molecular basis for this critical transition in the parasites life cycle is unknown. Six previously uncharacterized genes, Pfg14.744, Pfg14.745, Pfg14.748, Pfg14.763, Pfg14.752 and Pfg6.6 that are members of a 36 gene Plasmodium falciparum-specific subtelomeric superfamily were found to be expressed in parasites that are committed to sexual development as suggested by co-expression of Pfs16 and Pfg27. Northern blots demonstrated that Pfg14.744 and Pfg14.748 were first expressed before the parasites differentiated into morphologically distinct gametocytes, transcription continued to increase until stage II gametocytes were formed and then rapidly decreased. Immunofluorescence assays indicated that both proteins were only produced in the subpopulation of ring stage parasites that are committed to gametocytogenesis and both localized to the parasitophorous vacuole (PV)b of the early ring stage parasites. As the parasites continued to develop Pfg14.748 remained within the parasitophorous vacuole, while Pfg14.744 was detected in the erythrocyte. The 5' flanking region of either gene alone was sufficient to drive early gametocyte specific expression of green fluorescent protein (GFP). In parasites transfected with a plasmid containing the Pfg14.748 5' flanking region immediately upstream of GFP, fluorescence was observed in a small number of schizonts the cycle before stage I gametocytes were observed. This expression pattern is consistent with commitment to sexual differentiation prior to merozoite release and erythrocyte invasion. Further investigation into the role of these genes in the transition from asexual to sexual differentiation could provide new strategies to block malaria transmission. Microarray analysis was used to compare two clones derived from Plasmodium falciparum strain 3D7 parasites that differ in their ability to undergo gametocytogenesis. Clone G+ produces gametocytes and clone G- produces very few if any gametocytes. RNA was harvested from the cultures when the asexual parasitemia was 0.9-1.48% (day 4) (n=4) after setting up the gametocyte cultures and 5.2-5.58% (day 6) (n=4) prior to the appearance of morphologically distinct gametocytes and used to generate cDNA that was labeled with Cy3 or Cy5 and hybridized to the Plasmodium falciparum 70 mer oligonucleotide microarray developed by DeRisi and co-workers.