Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila’s life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface.

Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, we generate a paired transcriptome/proteome time course dataset with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (ρ = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, we postulate that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis and partially validate this hypothesis using Hrb98DE knockdown. In summary, we present a systems biology framework for the identification of post-transcriptional gene regulation from large-scale, time-resolved transcriptome and proteome data.

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although Plasmodium spp. genomes encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones upon heat-shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum . PfAP2-HS activates transcription of hsp70–1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70–1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37°C, but not at 35°C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or by the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.

BackgroundMalaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas.MethodsA cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-142 (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry.ResultsImmigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-142 (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria.ConclusionsUpon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.

The survival of malaria parasites in the changing human blood environment largely depends on their ability to alter gene expression by epigenetic mechanisms. The active state of Plasmodium falciparum clonally variant genes (CVGs) is associated with euchromatin characterized by the histone mark H3K9ac, whereas the silenced state is characterized by H3K9me3-based heterochromatin.

Lysine methylation is part of the posttranscriptional histone code employed to recruit modification specific readers to chromatin. Unbiased, quantitative mass spectrometry approaches combined with peptide pull-downs have been used to study histone methylation-dependent binders in mammalian cells. Here, we extend the study to birds by investigating the interaction partners for H3K4me3, H3K9me3, H3K27me3 and H3K36me3 in chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) using label-free quantitative proteomics. In general, we find very strong overlap in interaction partners for the trimethyl marks in birds compared to mammals, underscoring the known conserved function of these modifications. In agreement with their epigenetic role, we find binding of PHF2 and members of the TFIID, SAGA, SET1 and NURF complex to the activation mark H3K4me3. Our data furthermore supports the existence of a LID complex in vertebrates recruited to the H3K4me3 mark. The repressive marks are bound by the HP1 proteins and the EED subunit of the PRC2 complex as well as by WIZ. Like reported in the previous mammalian screens, we found ZNF462, ZNF828 and POGZ enriched at H3K9me3. However, we noted some unexpected differences. N-PAC (also known as GLYR1), an H3K36me3 interactor in mammals, is reproducible not enriched at this modification in our screen in birds. This initial finding suggests that despite strong conservation of the histone tail sequence, a few species-specific differences in epigenetic readers may have evolved between birds and mammals. All MS data have been deposited in the ProteomeXchange with identifier PXD002282 (http://proteomecentral.proteomexchange.org/dataset/PXD002282).

et al. (2020) SCIRT lncRNA restrains tumorigenesis by opposing transcriptional programs of tumor-initiating cells. Cancer Research.

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