CNOT1

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii , we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

The transmission of malaria parasites to mosquitoes is dependent on the formation of gametocytes. Once fully matured, gametocytes are able to transform into gametes in the mosquito's midgut, a process accompanied with their egress from the enveloping erythrocyte. Gametocyte maturation and gametogenesis require a well-coordinated gene expression program that involves a wide spectrum of regulatory proteins, ranging from histone modifiers to transcription factors to RNA-binding proteins. Here, we investigated the role of the CCCH zinc finger protein MD3 in Plasmodium falciparum gametocytogenesis. MD3 was originally identified as an epigenetically regulated protein of immature gametocytes and recently shown to be involved in male development in a barcode-based screen in P. berghei. We report that MD3 is mainly present in the cytoplasm of immature male P. falciparum gametocytes. Parasites deficient of MD3 are impaired in gametocyte maturation and male gametocytogenesis. BioID analysis in combination with co-immunoprecipitation assays unveiled an interaction network of MD3 with RNA-binding proteins like PABP1 and ALBA3, with translational initiators, regulators and repressors like elF4G, PUF1, NOT1 and CITH, and with further regulators of gametocytogenesis, including ZNF4, MD1 and GD1. We conclude that MD3 is part of a regulator complex crucial for post-transcriptional fine-tuning of male gametocytogenesis.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.