Biology subjects

Wohlschlegel, J.

Publications and source records attributed to Wohlschlegel, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual-Binding Scaffold RRsS1 Bridges Pol IV Transcripts and DNA Templates for Robust Reproductive siRNA Biogenesis

Cytosine DNA methylation established by RNA-directed DNA methylation (RdDM) is critical for transposon silencing and genome integrity in plants. In reproductive tissues, RNA Polymerase IV (Pol IV) is recruited to specific genomic loci by DNA motifs and the transcription factor-GDE1-CLSY3/4 complex to initiate abundant siRNA biogenesis and de novo DNA methylation, but the mechanisms that stabilize the Pol IV transcription complex at these sites remain elusive. Using immunoprecipitation-mass spectrometry of the Pol IV complex, we identify Reproductive RdDM siRNA Scaffold 1 (RRsS1) as a novel Pol IV interactor. RRsS1 colocalizes genome-wide with motif-dependent RdDM factors in floral tissues. Loss of RRsS1 significantly impairs 24-nt siRNA accumulation at a subset of CLSY3/4-dependent loci, particularly those exhibiting high Pol IV enrichment. We show that chromatin association of RRsS1 is stabilized by GDE1 and CLSY3/4. Structural modeling and biochemical assays characterize RRsS1 as a chimeric protein containing HRDC, Nudix, and DRBM modules that bind single-stranded DNA (ssDNA) and double-stranded DNA (dsRNA), respectively. Together, our findings support a model in which RRsS1 acts as a dual-binding scaffold, bridging Pol IV-RDR2 transcripts to their DNA templates within transcription bubbles to ensure robust siRNA production during plant reproduction.

plant biology↗

bioRxiv2026

J-domain proteins bridge the PKL and ATRX complex to transcription factors to fine-tune gene expression via H3.3 deposition

Epigenetic modifications, including histone modifications and DNA methylation, direct gene expression programs during the growth and development of eukaryotic organisms. In Arabidopsis thaliana, the chromatin remodeler PICKLE (PKL), a homolog of animal CHD3, plays a critical role during these processes. Previous studies of PKL have painted a complex picture of its function, including both activating and repressive roles, however, how PKL can control both transcriptional silencing and activation is unknown. We have identified a group of J-domain-containing proteins (DNAJs), usually known for their roles in protein folding, that guide PKL recruitment to various gene promoters by bridging PKL to transcription factors. Mutation of PKL's DNAJ-interacting domain disrupts PKL association with chromatin. Once recruited to transcription factor-bound sites, PKL coordinates with the histone chaperone ATRX to deposit HISTONE3.3 (H3.3) at targeted loci, which also negatively affects the accumulation of H3 lysine 27 trimethylation (H3K27me3) at promoters. We found that transcriptional outcome of PKL binding depends not only on the changes in H3.3 occupancy but also on the pre-existing chromatin context at PKL-targeted sites. Our findings outline a new mechanism for CHD3 chromatin remodeler recruitment and function.

plant biology↗

bioRxiv2026

A conserved vesicular circuit enables metabolic adaptation and maintains systemic heme homeostasis

Metabolic homeostasis depends on adaptive control of intracellular metabolite flux, yet how such control is reconfigured when canonical transport pathways fail is unknown. Here we define a conserved vesicular circuit that preserves systemic heme balance by rerouting intracellular heme flux. We show that loss of the intestinal heme exporter MRP-5 in Caenorhabditis elegans causes lethal heme sequestration within endolysosomal compartments. This defect is bypassed by disabling the vesicular adaptor AP-3, which stabilizes and reroutes the heme importer HRG-1, restoring heme export without increasing cytosolic heme. Unbiased genetics and transcriptomics identify two previously uncharacterized SLC49A family members, HRG-13 and HRG-14, as heme exporters with distinct affinities that engage in a vesicular importer-exporter handoff. Live imaging reveals heme-enhanced contacts between HRG-1 and SLC49A-containing vesicles, consistent with direct vesicular transfer. This circuitry extends to vertebrates as disruption of the SLC49A3 homolog impairs erythropoiesis in zebrafish and causes intracellular heme overload, premature hemoglobinization and apoptosis in differentiating human erythroid cells. Together, these findings establish SLC49A3 proteins as conserved heme exporters and uncover a general principle of metabolic adaptation in which reprogramming intracellular compartmentalization, rather than increasing nutrient supply, restores systemic homeostasis.

cell biology↗

bioRxiv2026

Stimulating neurogenesis of distinct retinal lineages in human retinal pigmented epithelium (RPE) with proneural transcription factors

There are currently few promising approaches for treatment of photoreceptor pathologies: for example, gene therapy to augment or replace mutated genes, has proven successful in preclinical studies, and some of these therapies are moving towards the clinic. Another approach aims to unlock the inherent stem-cell potential of non-neuronal retinal cells to regenerate neurons in situ. This line of research is based on the discovery that some vertebrates can restore even severely damaged retina from RPE with all the necessary cell types to regain full functionality. To determine whether this approach can be applied to humans, we established a robust in vitro culture system using fetal human RPE, and employed a barcode-multiplexed, single cell RNAseq based screen to find factors that would reprogram human RPE into a photoreceptors. With this approach we were able to identify NEUROD1 as a complimentary factor to ASCL1. Their combined overexpression together with a treatment with bFGF and Activin A inhibitor produced RPE-derived neuronal cells with expression patterns characteristic of photoreceptors and other lineages.

Developmental Biology↗

bioRxiv2026

Identification of a Putative Metal Transporter in the Apicoplast of Malaria Parasites

Plasmodium falciparum malaria parasites harbor an essential plastid organelle, called the apicoplast, which produces key metabolites required for organelle function and parasite viability. Apicoplast functions depend on iron and other metals, but the membrane transporters that mediate metal import into this organelle have been challenging to identify. Tetracycline antibiotics, including doxycycline, specifically target the apicoplast and can exhibit metal-dependent activity. Using tetracycline-affinity proteomics, we identified a doxycycline-interacting, uncharacterized transmembrane protein (UCT) targeted to the apicoplast periphery but not proteolytically processed. Although lacking sequence similarity to proteins of known function, UCT has a predicted structure with high similarity to pentameric CorA-family metal transporters that mediate metal uptake in other organisms. Functional tests revealed that UCT is dispensable for blood-stage asexual parasites, suggesting that the apicoplast has evolved redundant mechanisms for metal uptake. UCT knockdown in gametocytes, however, impairs the development of sexual parasites, which are critical for mosquito transmission. Our study identifies an apicoplast membrane protein with localization and structural properties that predict a role in metal transport into this key organelle. This discovery can provide a biochemical springboard to unravel broader apicoplast mechanisms of metal uptake across multiple stages of parasite development, including mosquito-stage parasites that display heightened UCT expression.

biochemistry↗

bioRxiv2026

The Cappuccino interactome reveals an intracellular role for Semaphorin-2a in Drosophila oogenesis

The spatiotemporal regulation of an actin mesh during Drosophila oogenesis is essential for proper localization of cell polarity determinants that establish the future patterning of the embryo. Here, we reveal an unexpected role for Semaphorin-2a (Sema2a) in actin mesh regulation and oogenesis. Sema2a classically functions as a secreted guidance cue that binds its cognate Plexin-B (PlexB) receptor to establish neural circuits. In contrast, we find that Sema2a is expressed inside the germarium, germline, and follicle cells of the developing ovary. Sema2a mutants possess small ovaries that fail to develop past mid-oogenesis. We demonstrate that Sema2a interacts with Cappuccino (Capu), a key actin nucleator crucial for building the actin mesh in Drosophila oocytes. Sema2a inhibits the actin assembly activity of Capu in vitro. Furthermore, genetic interaction between Sema2a and Capu influences mesh density and disrupts oskar mRNA localization. PlexB mutants, however, exhibit wild-type size ovaries with oskar mRNA localization distinct from Sema2a mutants, confirming the non-canonical role of Sema2a in oogenesis. SummaryThis study reveals a novel interaction between the actin nucleator Cappuccino and the typically secreted neural guidance factor Semaphorin-2a. It is shown that Semaphorin-2a inhibits the actin polymerization activity of Cappuccino in vitro and play an intracellular role in oogenesis.

cell biology↗

bioRxiv2026

PABPC1 Modulates Immunoglobulin pre-mRNA Alternative Polyadenylation

Alternative polyadenylation is a mechanism by which cells tune gene expression, and dysregulation can lead to development of disease. PABPC1 has been implicated in poly(A) site selection, but its function in gene regulation remains contradictory and poorly defined. Here, we investigate its role in B cell development, where APA controls immunoglobulin secretion. To define this role, we mapped PABPC1-RNA interactions using CLAP-seq and perturbed PABPC1 expression using a degron based strategy. PABPC1 localizes to the 3UTR in 70% of its gene targets and primarily binds to A-rich regions. Integration with transcriptomic data suggests PABPC1 downregulates 60% of its gene targets. While transcriptome-wide shifts in 3 UTR length were limited, PABPC1 binding was specifically enriched in genes exhibiting significant 3 UTR shortening. Using a foundational genomics model, we find the PAS-proximal region is the most predictive of gene expression within PABPC1 binding sites. Positional analysis revealed PABPC1 localizes closer to the PAS in genes downregulated following depletion. In immunoglobulin transcripts, PABPC1 binds to both secreted and membrane isoforms and is more enriched at the secretory PAS, and depletion modestly alters immunoglobulin expression. Together, our findings demonstrate PABPC1 primarily shortens and downregulates its targets in a context dependent manner. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/720383v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1a93633org.highwire.dtl.DTLVardef@257f55org.highwire.dtl.DTLVardef@1cad5bcorg.highwire.dtl.DTLVardef@19662fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Proximity proteomics reveals a role for IFI16 during human coronavirus infection

Viruses rely on the infected host cell to ensure successful replication and propagation of infection. This is achieved through interactions between virus-encoded proteins and proteins expressed in infected cells. All human coronaviruses (HCoVs) encode 16 non-structural proteins (NSPs) which exhibit some level of similarity in identity and function among the HCoVs. To identify host proteins that are potential interacting partners of HCoV NSPs, we utilized split-TurboID along with mass spectrometry and identified IFN-{gamma}-inducible protein-16 (IFI16) as a proximal partner of SARS-CoV-2 NSP8 and NSP10. To investigate the significance of the association between the NSP8/NSP10 complex and IFI16, we utilized CRISPR-Cas9 to knockout and CRISPRi to knockdown IFI16 in A549 cells and demonstrated that loss or reduced expression of IFI16 leads to a decrease in human coronavirus infection. We further demonstrated that there is reduced viral RNA replication and viral protein synthesis upon loss of IFI16. Interestingly, the loss of IFI16 results in reduced expression of type I IFNs. Taken together, these data suggests that IFI16 promotes human coronavirus infection, and the role IFI16 plays in coronavirus replication is independent of its role as a regulator of type I IFN gene expression.

microbiology↗

bioRxiv2026

TXNDC15 modulated quality control at the endoplasmic reticulum shapes ciliogenesis

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.

molecular biology↗

bioRxiv2026

The long noncoding RNA Malat1 contains an internal ribosome entry site mediating micropeptide translation

In most cells, Malat1 long noncoding RNA localizes to the nucleus where it affects splicing and chromatin function. In neurons Malat1 is exported to the cytoplasm where it is translated to generate the M1 micropeptide. Here we characterize an internal ribosome entry site (IRES) required for Malat1 translation. Although preceded by a long Malat1 5' RNA segment this element induces translation at the M1 AUG. In vivo chemical probing and structural modeling identified a 135 nt RNA secondary structure consisting of three stem loops that is sufficient for IRES activity. Using this minimal element for affinity purification from cell extracts, the IRES RNA selectively binds ribosomal subunits and translation factors. Depletion of the binding proteins Rack1 and hnRNP A2/B1 inhibits downstream IRES-dependent translation without affecting translation of an upstream ORF. Our study identifies an unexpected functional unit hidden within a widely studied long noncoding RNA.

molecular biology↗

bioRxiv2025

EBF1 regulates sensory establishment in the cochlea by positioning the medial boundary of the prosensory domain and restricting proliferation of the sensory progenitor population

In our previous study, we reported that Ebf1 excision throughout the inner ear epithelium and before the onset of cochlear development leads to dramatic sensory expansion in the cochlea by neonatal stages. Ebf1 conditional knockout cochleae possess over twice as many sensory cells as littermate controls and develop ectopic sensory patches in their Kollikers organs. To better understand the mechanism behind EBF1s role in restricting sensory establishment, we performed multiome sequencing in our current study. EBF1 is a transcription factor best known for its importance in B cell lineage specification, during which it acts as both an activator and a repressor. Our results indicate that EBF1 prevents the Kollikers organ cells from being recruited to the prosensory domain by promoting expression of Prdm16 and repressing expression of Jag1 and Sox2. We also found that EBF1 may promote cell cycle exit by repressing Ccnjl expression. In summary, medial expansion of the prosensory domain, together with delayed cell cycle exit in the developing cochlear epithelium, underlies the robust increase in sensory cells seen in Ebf1 conditional knockouts. SUMMARY STATEMENTMultiome sequencing reveals EBF1 regulates prosensory domain establishment by: (1) positioning the medial boundary of the prosensory domain and (2) restricting the proliferative capacity of the sensory progenitor pool.

developmental biology↗

bioRxiv2025

Dynamic expression of ASCL1 drives neurogenesis from infant and adult macaque Müller glia into immature retinal ganglion cells

Strategies to stimulate the regeneration of neurons in the adult central nervous system can offer universal solutions for neurodegenerative diseases. Taking lessons from naturally regenerating species, such as the zebrafish, we have previously shown that vector-mediated expression of proneural transcription factors can stimulate neurogenesis from the resident Muller glia (MG) population in the adult mouse retina, both in vitro and in vivo. To bring this closer to translation, we now show that vector-mediated expression of the proneural transcription factor ASCL1 can reprogram adult macaque MG into functional neurons. To this end, we established purified MG cultures and show they retain a mature transcriptomic profile that correlates with foveal and peripheral MG. Importantly, MG-derived neurons express retinal ganglion cell markers, can fire action potentials and have a transcriptome that overlaps with developing human and adult macaque retinal ganglion cells. To refine this approach for clinical application, we incorporated microRNA-124 target sites in the reprogramming cassette and show that this restricts expression to MG in mixed primary cultures and intact explant cultures of adult macaque retina. Regulating ASCL1 expression with microRNA-124 target sites maintained the reprogramming efficiency from adult MG cultures and improved the yield of RGC-like neurons from infant MG cultures. Most importantly, with this vector cassette we successfully reprogrammed macaque MG from both adult and infant retina into HuC/D+ neurons. Our findings demonstrate that ASCL1 can induce neurogenesis from macaque MG across ages and provide a targeted, effective strategy for potential clinical translation in retinal repair.

neuroscience↗

bioRxiv2025

Global Landscape of Human Kinase Motifs in Viral Proteomes

Viruses are classically viewed as targets of host sensing, yet whether they also sense and respond to host cues remains largely unexplored. We propose that host-driven post-translational modification of viral proteins allows viruses to dynamically sense host cellular states. We annotated human kinase motifs in 1,505 viral proteomes and discovered an enrichment for stress, inflammation, and cell-cycle kinases. Mapping kinase motifs onto 21,606 viral protein structures and integrating with phosphoproteomics of infected cells revealed surface-accessible residues were preferentially phosphorylated, showed greater kinase specificity, and were under positive selection for stress and immune kinase motifs. Temporal phosphoproteomics of alphavirus-infected cells confirmed stress kinase activation and viral protein phosphorylation, and MAP kinase inhibition reduced alphavirus replication and phosphorylation of ERK and JNK motifs on viral proteins. Our findings suggest that viruses evolved as biosensors of the host signaling state, unveiling new antiviral opportunities aimed at disrupting virus decision-making.

microbiology↗

bioRxiv2025

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

biochemistry↗

bioRxiv2025

Arabidopsis REM transcription factors and GDE1 shape the DNA methylation landscape through the recruitment of RNA Polymerase IV transcription complexes.

DNA methylation plays crucial roles in gene regulation and transposon silencing. In plants, the maintenance of DNA methylation is controlled by several self-reinforcing loops involving histone methylation and non-coding RNAs. However, how methylation is initially patterned at specific loci is unknown. Here, we describe four Arabidopsis REM transcription factors, VDD, VAL, REM12 and REM13, that recognize specific sequence regions, and together with the protein GENETICS DETERMINES EPIGENETICS1 (GDE1), recruit RNA polymerase IV transcription complexes to generate 24-nucleotide small interfering RNAs (24nt-siRNAs) that guide DNA methylation to specific loci. In the absence of GDE1, Pol IV transcription complexes redistribute to sites bound by a different factor called REM8. These results suggest that REM proteins act as sequence specific DNA binding proteins that pattern siRNAs and methylation at specific sites in the genome, highlighting the role of genetic information in determining epigenetic patterns.

plant biology↗

bioRxiv2024

Stimulating the regenerative capacity of the human retina with proneural transcription factors in 3D cultures

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Muller glia (MG) induces regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors such as ATOH1 and NEUROD1. Combining IHC, scRNA-seq and electrophysiology, we show for the first time that human MG can generate new neurons even in adults. This work constitutes a key step towards a future clinical application of this regenerative medicine approach for retinal degenerative disorders.

developmental biology↗

bioRxiv2024

Plant BCL-Domain Homologues play a conserved role in SWI/SNF complex stability

The SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are evolutionarily conserved, ATP-dependent chromatin remodelers crucial for multiple nuclear functions in eukaryotes. Recently, plant BCL-Domain Homolog (BDH) proteins were identified as shared subunits of all plant SWI/SNF complexes, significantly impacting chromatin accessibility and various developmental processes in Arabidopsis. In this study, we performed a comprehensive characterization of bdh mutants, revealing a previously overlooked impact on hypocotyl cell elongation. Through detailed analysis of BDH domains, we identified a plant-specific N-terminal domain that facilitates the interaction between BDH and the rest of the complex. Additionally, we uncovered the critical role of the BDH {beta}-hairpin domain, which is phylogenetically related to metazoan BCL7 SWI/SNF subunits. While phylogenetic analyses did not identify BDH/BCL7 orthologs in fungi, structure prediction modeling demonstrated strong similarities between the SWI/SNF catalytic modules of plants, animals, and fungi, and revealed the yeast Rtt102 protein as a structural homolog of BDH and BCL7. This finding is supported by the ability of Rtt102 to interact with the Arabidopsis catalytic module subunit ARP7 and partially rescue the bdh mutant phenotypes. Further experiments revealed that BDH promotes the stability of the ARP4-ARP7 heterodimer, leading to the partial destabilization of ARP4 in the SWI/SNF complexes. In summary, our study unveils the molecular function of BDH proteins in plant SWI/SNF complexes and suggests that {beta}-hairpin-containing proteins are evolutionarily conserved subunits crucial for ARP heterodimer stability and SWI/SNF activity across eukaryotes.

plant biology↗

bioRxiv2024

Ciliary marginal zone of the developing human retina maintains retinal progenitor cells until late gestational stages

Non-mammalian vertebrates maintain a proliferative stem cell population at the far periphery of their retina called the ciliary marginal zone (CMZ), which gives rise to all retinal cell types and contributes to retinal regeneration upon injury. Humans do not maintain a proliferative CMZ into adulthood; however, it is not known how long in development this region continues to generate new neurons. Here, we identify a population of cells in the far peripheral retina of the fetal human that continues to proliferate long after the rest of the retina is quiescent. Single cell RNA-sequencing and EdU tracing at late time points in development reveal that this region has features of the non-mammalian CMZ, including the capacity to produce both early and late born cell types at late developmental stages, and a longer cell cycle than more centrally located retinal progenitor cells (RPCs). Moreover, while more central RPCs exit the cell cycle with the addition of a TGF{beta}-inhibitor, we show that early RPCs within the CMZ do not. These findings define the late stages of neurogenesis in human retinal development, and present a unique model system to study the fetal CMZ in humans.

developmental biology↗