Biology subjects

Jami-Alahmadi, Y.

Publications and source records attributed to Jami-Alahmadi, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Acyl Carrier Protein is Essential for Apicoplast Biogenesis in Malaria Parasites Independent of Fatty Acid Synthesis

Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type-II fatty acid synthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for P. falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In contrast to FASII enzymes that can be disrupted without fitness defect, we report that knockout or ligand-dependent knockdown of apicoplast ACP is lethal to blood-stage P. falciparum, indicating an essential FASII-independent function. Loss of ACP impairs the biosynthesis of essential isoprenoid precursors and blocks apicoplast biogenesis. Using proximity biotinylation and biochemical interaction studies, we identified a key role for ACP in binding and stabilizing apicoplast pyruvate kinase II (PKII). This critical enzyme is the only known source of nucleoside triphosphates (NTPs) in this organelle and is required for isoprenoid synthesis and apicoplast biogenesis. Our work reveals that ACP knockdown results in destabilization and loss of PKII, which is sufficient to explain ACP essentiality in this stage. This work unveils essential ACP function at a key biochemical hub controlling broad apicoplast metabolism in malaria parasites that is independent of the canonical ACP role in FASII.

biochemistry↗

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2024

EDC-3 and EDC-4 Regulate Embryonic mRNA Clearance and Biomolecular Condensate Specialization

Animal development is dictated by the selective and timely decay of mRNAs in developmental transitions, but the impact of mRNA decapping scaffold proteins in development is unknown. This study unveils the roles and interactions of the DCAP-2 decapping scaffolds EDC-3 and EDC-4 in the embryonic development of C. elegans. EDC-3 facilitates the timely removal of specific embryonic mRNAs, including cgh-1, car-1, and ifet-1 by reducing their expression, and preventing excessive accumulation of DCAP-2 condensates in somatic cells. We further uncover a novel role for EDC-3 in defining the boundaries between P-bodies, germ granules, and stress granules. Lastly, we show that EDC-4 counteracts EDC-3 and engenders the assembly of DCAP-2 with the GID (CTLH) complex, a ubiquitin ligase involved in maternal-to-zygotic transition (MZT). Our findings support a model wherein multiple RNA decay mechanisms temporally partake in the clearance of maternal and zygotic mRNAs throughout embryonic development.

molecular biology↗

bioRxiv2024

SCF FBXL5 ubiquitin ligase regulates stability of von-Hippel Lindau protein and the HIF1α-dependent response to hypoxia

The canonical response to changes in cellular oxygen levels consists of the ubiquitin-dependent degradation of hypoxia-inducible transcription factors (HIFs) in a prolyl hydroxylase (PHD) and von-Hippel Lindau-ElonginB-ElonginC (VHL-ElonginBC) E3 ubiquitin ligase complex-dependent manner. This regulated degradation event is oxygen-dependent and results in activation of a transcriptional program that mediates the cellular adaptation to changes in oxygen tension. Here, we show that a distinct Cullin-RING ligase complex, SKP1-CUL1-FBXL5 (SCFFBXL5), physically associates with VHL and promotes its ubiquitin-dependent degradation during hypoxia. The regulation of VHL protein stability by FBXL5 influences HIF1 expression levels and the transcriptional activation of downstream hypoxia-responsive target genes. This work identifies a novel mechanism for VHL regulation which contributes to the HIF1-mediated cellular response to hypoxia and provides an additional layer of crosstalk between iron and oxygen homeostasis.

molecular biology↗

bioRxiv2024

Microglia promote extracellular matrix deposition and restrict excitatory synapse numbers in the mesolimbic dopamine system during healthy aging

Synapse dysfunction is tightly linked to cognitive changes during aging. Emerging evidence suggests that microglia and the extracellular matrix (ECM) can potently regulate synapse integrity and plasticity. Yet the brain ECM, and its relationship with microglia, synapses, and cognition during aging remains virtually unexplored. Using ECM-optimized proteomic workflows and histological analyses, we discovered striking regional differences in ECM composition and aging-induced ECM remodeling across key basal ganglia nuclei. Moreover, we combine two distinct behavioral classification strategies with fixed-tissue confocal imaging and proteomic analysis to identify robust relationships between the hyaluronan- and proteoglycan-rich ECM and cognitive aging phenotypes. Finally, we provide evidence that aging midbrain microglia lose capacity to interact with and regulate the ECM, and that these aging-associated microglial changes are accompanied by local ECM accumulation and worse behavioral performance. Together, these foundational observations implicate changing microglia-ECM-synapse interactions as a key determinant of cognitive functioning during healthy aging.

neuroscience↗

bioRxiv2023

The apoptotic splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

molecular biology↗

bioRxiv2023

ACD15, ACD21, and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: -crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies. One-Sentence SummaryArabidopsis ACD21 and ACD15 drive accumulation of MBD5/6 complex silencing assemblies at methyl-CG sites and recruit SLN to maintain protein mobility in these assemblages.

cell biology↗

bioRxiv2023

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels.

Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

microbiology↗

bioRxiv2023

Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

microbiology↗

bioRxiv2023

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation.

MOM1 is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we found that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with artificial zinc finger to unmethylated FWA promoter led to establishment of DNA methylation and FWA silencing. This process was blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MORC6. We found that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes was impaired by mutation of MOM1 or mutation of genes encoding the MOM1 interacting PIAL1/2 proteins. In addition to RdDM sites, we identified a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.

plant biology↗

bioRxiv2023

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

plant biology↗

bioRxiv2022

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis

DNA methylation has been utilized for target gene silencing in plants, however its not well-understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain of function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression either through the establishment of DNA methylation, or via DNA methylation-independent processes including histone H3K27me3 deposition, H3K4me3 demethylation, H3K9, H3K14, H3K27, and H4K16 deacetylation, inhibition of RNA Polymerase II transcription elongation or Ser-5 dephosphorylation. The silencing fusion proteins also silenced many other genes with different efficacy, and a machine learning model could accurately predict the efficacy of each silencer based on various chromatin features of the target loci. These results provide a more comprehensive understanding of epigenetic regulatory pathways and provide an armament of tools for targeted manipulation of gene expression.

plant biology↗

bioRxiv2022

MORC proteins regulate transcription factor binding by mediating chromatin compaction in active chromatin regions

BackgroundThe Microrchidia (MORC) proteins are a family of evolutionarily conserved GHKL-type ATPases involved in chromatin compaction and gene silencing. Arabidopsis MORC proteins act in the RNA-directed DNA methylation (RdDM) pathway, where they act as molecular tethers to ensure the efficient establishment of RdDM and de novo gene silencing. However, MORC proteins also have RdDM-independent functions; although, their underlying mechanisms are unknown. ResultsIn this study, we examined regions of MORC binding where RdDM does not occur in order to shed light on the RdDM-independent functions of MORC proteins. We found that MORC proteins compact chromatin and reduce DNA accessibility to transcription factors (TFs), thereby repressing gene expression. We also found that MORC-mediated repression of gene expression was particularly important under conditions of stress. We showed that MORC proteins regulate TFs through either direct or indirect interactions, and these TFs can in some cases regulate their own transcription, resulting in feedforward loops. ConclusionsOur findings provide insights into the molecular mechanisms of MORC-mediated chromatin compaction and transcription regulation.

plant biology↗

bioRxiv2022

Multi-site phosphorylation of the TOPBP1 ATR Activation Domain propels ATR signaling during the response to DNA breaks

TOPBP1 is a BRCT domain-containing scaffold protein that plays important roles in a diverse array of cellular processes, including DNA damage signaling, DNA repair, DNA replication, and mRNA transcription. For DNA damage signaling, TOPBP1 activates the crucial damage response kinase ATR and this occurs during replication stress as well as during a DNA double-strand break response. ATR signaling allows cells to survive genotoxic issues and represents a formidable barrier to transformation to a tumorigenic state. Despite its importance to genome stability, the biochemical mechanism for how TOPBP1 activates ATR is not fully understood. TOPBP1 uses a discrete domain, termed the ATR activation domain, to stimulate ATR kinase. Recent work has shown that the AAD must be in a multimeric state to activate ATR. Other work has shown that phosphorylation of the AAD on serine 1131 (in Xenopus) is important for its function, and some have suggested that this is linked to the formation of TOPBP1 condensates during a DNA damage response. In this study we examine AAD phosphorylation in detail and we report three important new findings. One, S1131 phosphorylation promotes ATR activation in a manner independent of condensate formation and is instead linked to promoting multimerization of the AAD. Two, we identify a novel sight of AAD phosphorylation, on T1098, and show that is required for ATR activation. Three, we identify additional, candidate phosphorylation sites, some of which fit the consensus for casein kinase 2, and we show that casein kinase 2 activity is required for the AAD to perform its function. These studies show that multi-site phosphorylation of the AAD is an important component of the mechanism by which TOPBP1 activates ATR.

cell biology↗

bioRxiv2022

Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.

plant biology↗

bioRxiv2022

Transcriptional drifts associated with environmental changes in endothelial cells

O_LIEnvironmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA-seq and protein expression by LC-MS directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interations by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We identify novel flow depedent genes, as well as, genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information and physical forces. C_LI

cell biology↗

bioRxiv2022

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and 5' transcript length determination in Arabidopsis.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of the plant BAF-like complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcriptional start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5 transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and 5 transcript length determination.

molecular biology↗

bioRxiv2022

The Nse5/6-like SIMC1-SLF2 Complex Localizes SMC5/6 to Viral Replication Centers

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1s Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1s helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions, binds SLF2 analogously to SIMC1 and forms a distinct Nse5/6-like complex. Thus, two Nse5/6-like complexes independently regulate human SMC5/6: SIMC1-SLF2 responding to viral challenge and SLF1/2 recognizing DNA damage.

cell biology↗