The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum.

BackgroundGene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium's genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum.ResultsClose to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p < 0.0001). Among the experimentally validated mRBPs are many known translational regulators active in other stages of the parasite's life cycle, such as DOZI, CITH, PfCELF2, Musashi, and PfAlba1-4. Finally, we also detect several proteins with an RNA-binding domain abundant in Apicomplexans (RAP domain) that is almost exclusively found in apicomplexan parasites.ConclusionsCollectively, our results provide the most complete comparative genomics and experimental analysis of mRBPs in P. falciparum. A better understanding of these regulatory proteins will not only give insight into the intricate parasite life cycle but may also provide targets for novel therapeutic strategies

The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum.

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies

Yeast Sgf73/Ataxin-7 serves to anchor the deubiquitination module into both SAGA and Slik(SALSA) HAT complexes

Spinocerebellar ataxia (SCA) is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1–3, 6–10, 12, and 17 result from a trinucleotide repeat expansion in the DNA-coding sequence. Intriguingly, recent work has demonstrated that increased trinucleotde expansions in the SCA7 gene result in defect in the function of the SAGA histone acetyltransferase complex. The SCA7 gene encodes a subunit of the SAGA complex. This subunit is conserved in yeast as the SGF73 gene. We demonstrate that Sgf73 is required to recruit the histone deubiquitination module into both SAGA and the related SliK(SALSA) complex, and to maintain levels of histone ubiquitination, which is necessary for regulation of transcription at a number of genes

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins

<p>Laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.</p

A Mammalian Mediator Subunit that Shares Properties with Saccharomyces cerevisiae Mediator Subunit Cse2

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193open reading frame. In this study, we present direct biochemical evidence that the FLJ10193protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex

The ATAC Acetyltransferase Complex Coordinates MAP Kinases to Regulate JNK Target Genes

SummaryIn response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling

The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that the two components of the SF3B complex, SF3B3 and SF3B5, that form part of the U2 small nuclear ribonucleoprotein particle (snRNP) are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5mutant flies, we show that SF3B5 is necessary for proper development and cell viability but not for histone acetylation. Although SF3B5 does not appear to function in SAGA\u27s histone-modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA\u27s transcription coactivator activity that is separate from its role in splicing

Regulation of the CRL4(Cdt2) ubiquitin ligase and cell-cycle exit by the SCF(Fbxo11) ubiquitin ligase

F-box proteins and DCAF proteins are the substrate binding subunits of the Skp1-Cul1-F-box protein (SCF) and Cul4-RING protein ligase (CRL4) ubiquitin ligase complexes, respectively. Using affinity purification and mass spectrometry, we determined that the F-box protein FBXO11 interacts with CDT2, a DCAF protein that controls cell-cycle progression, and recruits CDT2 to the SCF(FBXO11)complex to promote its proteasomal degradation. In contrast to most SCF substrates, which exhibit phosphodegron-dependent binding to F-box proteins, CDK-mediated phosphorylation of Thr464 present in the CDT2 degron inhibits recognition by FBXO11. Finally, our results show that the functional interaction between FBXO11 and CDT2 is evolutionary conserved from worms to humans and plays an important role in regulating the timing of cell-cycle exit.Fil: Rossi, Mario. University Of New York; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Duan, Shanshan. University Of New York; Estados Unidos. Howard Hughes Medical Institute; Estados UnidosFil: Jeong, Yeon Tae. University Of New York; Estados UnidosFil: Horn, Moritz. Max Planck Institute for Biology of Ageing; Alemania. University of Cologne; AlemaniaFil: Saraf, Anita. The Stowers Institute for Medical Research; Estados UnidosFil: Florens, Laurence. The Stowers Institute for Medical Research; Estados UnidosFil: Washburn, Michael P.. The Stowers Institute for Medical Research; Estados Unidos. University of Kansas; Estados UnidosFil: Antebi, Adam. Max Planck Institute for Biology of Ageing; Alemania. University of Cologne; AlemaniaFil: Pagano, Michele. University Of New York; Estados Unidos. Howard Hughes Medical Institute; Estados Unido

Determining Protein Complex Connectivity Using a Probabilistic Deletion Network Derived from Quantitative Proteomics

Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex