Biology subjects

Busch, A.

Publications and source records attributed to Busch, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Granule microenvironment regulates the dual functions of FMR1

Fragile X Messenger Ribonucleoprotein 1 (FMR1) is an evolutionarily conserved RNA binding protein with important functions in cognition and female reproduction, and its disruption is associated with neurodevelopmental and reproductive disorders including the Fragile X syndrome. FMR1 is best known for its role as a translation repressor. However, several recent studies also suggest a role of FMR1 as a translation enhancer raising fundamental questions about the molecular regulation of these opposing functions. In this study, we identify FMR1 as part of the oskar mRNA-protein complex in the Drosophila oocyte and study the role of FMR1 as a translational enhancer of oskar. We provide the molecular mechanism for the dual functions of FMR1 and show that the two major RNA-binding domains of FMR1, the KH domains and the RGG box, play distinct separable roles in regulating translation. The KH domains enhance translation of mRNAs while the RGG box containing C-terminal domain (CTD) is required to repress translation. We further show that the condensation propensity of FMR1 containing granules regulates the two antagonistic functions, such that phase separation by FMR1-CTD creates the molecular microenvironment necessary for the repressive activity, whereas reduction in phase separation is associated with increased translation. Our findings highlight the importance of biomolecular condensates not just as a means of molecular compartmentalization but as a fundamental regulatory principle that dictates the functional output of modular protein domains.

molecular biology↗

bioRxiv2026

Phase separation behavior of TDP-43 governs its protein interactome and regulation of altern

TDP-43 is a nuclear RNA-binding protein that regulates RNA metabolism, including alternative splicing. Its aggregation is a major pathological hallmark of several neurodegenerative diseases. TDP-43 undergoes phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43 RNA regulatory functions remains poorly understood. Here we utilized rationally designed mutations in the TDP-43 low complexity domain to tune TDP-43 PS, yielding a panel of TDP-43 variants with reduced propensity to form condensates (PS-deficient), and a panel forming irreversible, undynamic condensates (solid-like) in vitro and in cells. Two complementary interactomics approaches identified PS-dependent interactions between TDP-43 and key RNA regulatory factors, including splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like variants. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis by modulating a subset of TDP-43-dependent alternative splicing events and by reshaping interactions with RNA regulatory factors.

biochemistry↗

bioRxiv2026

PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

The DNA damage response (DDR) is a complex network of interconnected pathways and sub-pathways that safeguards genome integrity. Deciphering the coordinated and complementary interactions among these pathways remains a major challenge. In this study, we employed CRISPR screening to systematically map the genetic interactions required for different sub-pathways of homologous recombination in human cells following PARP inhibitor treatment. Our approach recapitulated known interactions and uncovered several previously unrecognized connections. We identified RAD54L, in addition to ATRX, as a factor promoting the double Holliday junction (dHJ) pathway and demonstrated that RAD51AP1 and RAD54B function in synthesis-dependent strand annealing (SDSA). We provide evidence that loss of TOP3A induces a switch in HR sub-pathway usage from SDSA to the dHJ pathway. Furthermore, TOP3A deficiency abolishes the requirement for ATRX and the histone variant H3.3 in the dHJ pathway, while maintaining strict dependence on RAD54L. We further observed that H3.3 is involved in both HR sub-pathways, whereas its depositing chromatin remodelers HIRA and ATRX play pathway-specific roles in SDSA and dHJ, respectively. Together, our findings define the architecture underlying HR sub-pathway choice and reveal a key role for TOP3A in regulating pathway balance.

molecular biology↗

bioRxiv2026

iCLIP3: A streamlined, non-radioactive protocol for mapping protein-RNA interactions in cellular transcripts at single-nucleotide resolution

UV-C crosslinking and immunoprecipitation (CLIP)-based methods are the gold standard for identifying direct RNA binding protein (RBP) interaction sites on cellular RNA in vivo. Here, we describe individual-nucleotide resolution CLIP version 3 (iCLIP3), an optimized protocol for generating transcriptome-wide maps of RBP-RNA interaction sites at single-nucleotide resolution from low-input material. iCLIP3 introduces several key improvements over previous iCLIP variants, including rapid and safe infrared-based visualization of RBP-RNA complexes, silica column-based RNA isolation, and the incorporation of TruSeq adapter sequences with unique dual indexing. These modifications streamline library preparation, facilitate multiplexing, and enable concurrent sequencing of iCLIP3 libraries alongside unrelated RNA-seq libraries. In addition, we provide a detailed bioinformatics workflow for identifying crosslinking events and defining RBP binding sites. The complete protocol can be performed within 4-5 days, offering a robust and efficient approach for high-resolution mapping of RBP-RNA interactions. HighlightsO_LI3' RNA labeling with pCp-IR750 enables rapid infrared visualization of RBP-RNA complexes C_LIO_LISilica column-based RNA isolation improves efficiency and reproducibility C_LIO_LITruSeq adapters with unique dual indexing enable flexible and scalable multiplexing C_LIO_LIiCLIP3 generates high-quality libraries from ultra-low amounts of cellular material C_LI

genomics↗

bioRxiv2026

RNA G-quadruplexes mediate cooperativity in HNRNPH binding and splicing regulation

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer. HighlightsO_LIHundreds of cassette exons are cooperatively regulated by HNRNPH. C_LIO_LIUnfolding of RNA G-quadruplexes (rG4s) at HNRNPH binding sites facilitates indirect cooperativity in RNA binding. C_LIO_LIMulti-step splicing amplifies the response into highly switch-like regulation. C_LIO_LIrG4-disrupting variants and changing HNRNPH expression are associated with breast cancer phenotypes. C_LI

molecular biology↗

bioRxiv2026

SLBP-independent control of maternal histone mRNA

Replication-dependent (RD) histones are crucial for packaging newly replicated DNA into chromatin, ensuring genome stability. In metazoans, the mRNA of RD histones is uniquely regulated through a conserved 3' stem-loop bound by stem-loop binding protein (SLBP). This allows cell cycle-coupled regulation of these important transcripts. However, oocytes must stabilise histone mRNAs independently of the cell cycle to ensure maternal loading to support the first embryonic divisions. Using Caenorhabditis elegans as a model system, we discovered an SLBP-independent mechanism that ensures RD histone transcript stability during oogenesis. This is mediated by the protein complex PETISCO, bound to the effector protein TOST-1, which directly binds the histone stem-loop region and maintains maternal histone mRNA levels during oogenesis and early embryogenesis. Loss of this mechanism disrupts histone homeostasis, leading to premature genome activation, mitotic defects, and embryonic lethality. Interestingly, the same complex, PETISCO, acts in piRNA biogenesis when bound to the effector PID-1, revealing an intriguing co-option of this histone mRNA homeostasis mechanism by the piRNA pathway. Our findings reveal a unique SLBP-independent mechanism of histone mRNA regulation, that served as a basis for the evolution of a novel piRNA biogenesis mechanism.

molecular biology↗

bioRxiv2025

Quantitative metagenomics using a portable protocol.

A field-deployable DNA sequencing approach for quantitative microbial community profiling can enable rapid responses for a range of applications in the water sector - from process control to wastewater surveillance. Current quantitative approaches have complex instrumentation requirements and long turnaround time for DNA recovery and absolute quantitation. In this study, we report a field-deployable rapid detection and absolute quantitation (rD+rQ) workflow that leverages the real-time sequencing capabilities of Nanopore sequencing for quantitative metagenomics. This workflow integrates a high-molecular-weight DNA recovery protocol for diverse environmental matrices of relevance to the water sector and multiplexed Nanopore sequencing with barcoded spike-in-based calibration (BSINC). BSINC using multi-species genomic spike-in controls exhibits significantly higher calibration accuracy compared to conventional approaches that utilize either single DNA fragment or single organism spike in controls. Dynamic detection and quantitation limits were established based on coverage fraction of sequenced genomes and the coefficient of variation of genome copy numbers across replicates to enhance the accuracy and precision of microbial quantitation. The rD+rQ workflow achieves species-level identification and absolute quantitative results comparable to digital PCR in environmental samples. This portable laboratory and easy-to-use rD+rQ workflow should facilitate rapid decision-making for the water industry.

microbiology↗

bioRxiv2025

Dissecting the RNA binding capacity of the multi-RRM protein Rrm4 essential for endosomal mRNA transport

RNA-binding proteins (RBPs) utilize multiple RNA-binding domains (RBDs) to engage with extensive mRNA networks. Understanding the intricate interplay of modular RBDs is essential for uncovering RBP function. Yet, how individual RBDs shape transcriptome-wide interactions remains poorly understood. Here, we dissect the roles of the three RNA recognition motifs (RRMs) in the endosomal mRNA transporter Rrm4 during polar growth of Ustilago maydis. Using a comparative mutant-based iCLIP2 approach, we disclose an extensive inventory of RRM-specific binding sites. Most binding sites are prominently governed by RRM3, however, they are not critical for function. Conversely, functionally essential binding sites are recognized by a more complex RBD interplay, involving RRM1 and/or RRM2 with partial support from RRM3. By integrating transcriptome-wide RNA binding data with transcriptomics, we pinpoint their function as regulatory RNA elements affecting mRNA abundance, linking endosomal transport to stability. The modular RNA binding of Rrm4 defines distinct RNA regulons controlling mitochondrial activity, polarity factors, and cell wall remodeling - processes critical for polar growth. These findings disclose the intricate binding modes of an RBP in vivo, emphasizing how multiple RBDs differentiate functional binding sites from accessory ones to determine mRNA fate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/636894v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1af77b6org.highwire.dtl.DTLVardef@dc7178org.highwire.dtl.DTLVardef@9d7ebforg.highwire.dtl.DTLVardef@1ec5a05_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Coordinated alternative splicing decisions via stepwise exon definition

Alternative splicing of pre-mRNA is a fundamental step in human gene regulation and aberrant slic-ing is tightly linked to diseases including cancer. Various splicing events, such as alternative exon (AE) choice and intron retention (IR), are controlled by a common molecular machinery, the spliceo-some. However, it remains elusive how the regulation of spliceosome activity coordinately affects these splicing decisions. Here, we analyze a large-scale mutagenesis screen and genome-wide RNA sequencing data to show that IR and AE choice are tightly coupled, as IR products primarily accu-mulate in alternative exons showing intermediate inclusion levels. Using data-driven mathematical modeling, we reveal that multistep exon recognition by the spliceosome explains the observed AE-IR dependency for cis-acting sequence mutations and upon knockdown of trans-acting RNA-binding proteins. Furthermore, we show that multistep exon definition is frequently perturbed in cancer cells, which leads to the coordinated deregulation of intron retention and AE choice. In conclusion, we showed that the spliceosome coordinates complex splicing decisions via stepwise exon definition, which potentially facilitates the search for common molecular mechanisms for mis-splicing in cancer.

systems biology↗

bioRxiv2024

Integrating Machine Learning with Flow-Imaging Microscopy for Automated Monitoring of Algal Blooms

Real-time monitoring of phytoplankton in freshwater systems is critical for early detection of harmful algal blooms so as to enable efficient response by water management agencies. This paper presents an image processing pipeline developed to adapt ARTiMiS, a low-cost automated flow-imaging device, for real-time algal monitoring specifically in freshwater and environmental systems. This pipeline addresses several challenges associated with autonomous imaging of aquatic samples such as flow-imaging artifacts (i.e., out-of-focus and background objects), as well as specific challenges associated with monitoring of open environmental systems (i.e., identification of novel objects). The pipeline leverages a Random Forest model to identify out- of-focus particles with an accuracy of 89% and a custom background particle detection algorithm to identify and remove particles that erroneously appear in consecutive images with >97{+/-}2.8% accuracy. Furthermore, a convolutional neural network (CNN), trained to classify distinct classes comprising both taxonomical and morphological categories, achieved 94% accuracy in a closed dataset. Nonetheless, the supervised closed-set classifiers struggled with the accurate classification of objects when challenged with debris and novel particles which are common in complex open environments; this limits real-time monitoring applications by requiring extensive manual oversight. To mitigate this, three methods incorporating classification with rejection were tested to improve model precision by excluding irrelevant or unknown classes. Combined, these advances present a fully integrated, end-to-end solution for real-time HAB monitoring in open environmental systems thus enhancing the scalability of automated detection in dynamic aquatic environments. HighlightsO_LIRandom Forest model is more generalizable than Convolutional Neural Networks to remove out-of-focus particles. C_LIO_LIA two-stage clustering algorithm is effective at removing background particles in flow imaging microscopy. C_LIO_LIClosed-set CNN classifier performance deteriorates when challenged with unknown particles. C_LIO_LIClassification with rejection improves both precision and accuracy for environmental samples. C_LI

ecology↗

bioRxiv2024

Microbial iCLIP2: Enhanced mapping of RNA-Protein interaction by promoting protein and RNA stability

The entire RNA lifecycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV crosslinking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges such as inherently high RNase and protease activity in fungi were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of crosslink events and improved the identification of Rrm4 binding sites. Thus, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complex I, III and V - revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study serves as a paradigm for optimizing iCLIP2 procedures in challenging organisms or tissues under high RNase/ protease conditions.

molecular biology↗

bioRxiv2022

Utilization of an Artery-on-a-chip to unravel novel regulators and therapeutic targets in vascular diseases

IntroductionOrgans-on-chips represent novel in vitro models that have the capacity to emulate aspects of human physiology and pathophysiology by incorporating features like tissue-multicellularity and exposure to organ-relevant physical environment. We developed an artery-on-a-chip with the objective to recapitulate the structure of the arterial wall composed of intimal and medial layers and the relevant hemodynamic forces that affect luminal cells. ResultsBy comparing arteries-on-chips exposed either to in vivo-like shear stress values or kept in static conditions, we identified a panel of novel genes modulated by shear stress. We next measured the expression pattern of shear stress-modulated genes in areas of the vascular tree affected by atherosclerotic plaques and aortic aneurysms, where disease development and progression are induced by alterations of shear stress. We obtained biopsies from patients affected by carotid artery disease (CAD), comprising the atherosclerotic plaque (diseased artery) and the adjacent region (non-diseased artery). From patients with abdominal aortic aneurysms (AAA), we obtained the aneurysmal portion (diseased aorta) and non-dilated adjacent segment (non-diseased aorta). Genes modulated by shear stress followed the same expression pattern in non-diseased segments of human vessels and were expressed by endothelial and smooth muscle cells as evidenced by immunofluorescence analysis and single cell RNA sequencing. Using mice and porcine models of vascular CAD and AAA, we confirmed that shear stress mediated targets are important in discriminating diseased and non-diseased vessel portions in vivo. Furthermore, we showed that our artery-on-a-chip can serve as a platform for drug-testing. We were able to reproduce the effects of a therapeutic agent previously used in AAA animal models in artery-on-a-chip systems and extend our understanding of its therapeutic effect through a multicellular structure. ConclusionsOur novel in vitro model is capable of mimicking important physiological aspects of human arteries, such as the response to shear stress, and can further shed light on the mechanism of action of potential therapeutics before they enter the clinical stage. TeaserThe artery-on-a-chip is a novel in vitro platform that enables the mimicry of human arteries and can be used to gain insights into the development and therapeutic targeting of vascular diseases.

cell biology↗

bioRxiv2022

Targeting long non-coding RNA NUDT6 enhances smooth muscle cell survival and limits vascular disease progression

Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target Fibroblast Growth Factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal models of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases.

molecular biology↗

bioRxiv2022

The circular RNA Ataxia-telangiectasia mutated (cATM) regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is often accidental, and for now, the only available treatment option remains surgical intervention. Circular RNAs (circRNAs) are RNA loops that originated from backsplicing, which have received increasing attention as a novel class of functional non-coding RNAs contributing to cardiovascular physiology and disease. Their high structural stability, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We aimed to investigate the contribution of circRNAs to AAA pathogenesis and their potential application as biomarkers for AAA diagnosis. Combined circRNA array and quantitative real-time PCR analysis revealed the presence of differentially expressed circular transcripts stemming from AAA-relevant gene loci. Among these, the circRNA to the Ataxia-telangiectasia mutated gene (cATM) was upregulated in human AAA tissue specimens, in AAA patient-derived SMCs, and serum samples collected from aneurysm patients. In control primary aortic SMCs, cATM increased upon angiotensin II stimulation, while its silencing triggered apoptosis. Furthermore, doxorubicin could induce cATM expression, supporting a link with acute stress response in SMCs. Constitutively higher cATM expressing AAA patient-derived SMCs were less vulnerable to oxidative stress-induced cell death and survival pathways enriched when compared to control SMCs. Taken together, this data supports the role of cATM in adapting SMCs to oxidative stress in the vascular AAA micromilieu. This molecular signature provides an additional parameter to be included in procedures for AAA screening in combination with already established practices.

molecular biology↗

bioRxiv2022

Neural sequences in primate prefrontal cortex encode working memory in naturalistic environments

Working memory is the ability to briefly remember and manipulate information after it becomes unavailable to the senses. The mechanisms supporting working memory coding in the primate brain remain controversial. Here we demonstrate that microcircuits in layers 2/3 of the primate lateral prefrontal cortex dynamically represent memory content in a naturalistic task through sequential activation of single neurons. We simultaneously recorded the activity of hundreds of neurons in the lateral prefrontal cortex of macaque monkeys during a naturalistic visuospatial working memory task set in a virtual environment. We found that the sequential activation of single neurons encoded trajectories to target locations held in working memory. Neural sequences were not a mere successive activation of cells with memory fields at specific spatial locations, but an abstract representation of the subjects trajectory to the target. Neural sequences were less correlated to target trajectories during perception and were not found during working memory tasks lacking the spatiotemporal structure of the naturalistic task. Finally, ketamine administration distorted neural sequences, selectively decreasing working memory performance. Our results indicate that neurons in the lateral prefrontal cortex causally encode working memory in naturalistic conditions via complex and temporally precise activation patterns.

neuroscience↗

bioRxiv2022

TERRA increases at short telomeres in yeast survivors and regulates survivor associated senescence (SAS)

Cancer cells achieve immortality by employing either homology-directed repair (HDR) or the telomerase enzyme to maintain telomeres. ALT (alternative lengthening of telomeres) refers to the subset of cancer cells that employ HDR. ALT mechanisms are strongly conserved from yeast to human cells, with the yeast equivalent being referred to as survivors. The non-coding RNA, TERRA and its ability to form RNA-DNA hybrids have been implicated in ALT/survivor maintenance by promoting HDR. It is not understood which telomeres in ALT/survivors engage in HDR, nor is it clear which telomeres upregulate TERRA. Using yeast survivors as a model for ALT, we demonstrate that HDR only occurs at telomeres when they become critically short. Moreover, TERRA levels steadily increase as telomeres shorten and decrease again following HDR-mediated recombination. Surprisingly, we observe that survivors undergo cycles of senescence, in a similar manner to non-survivors following telomerase loss, which we refer to as survivor associated senescence (SAS). Similar to "normal" senescence, we observe that RNA-DNA hybrids slow the rate of SAS by decreasing the rate of telomere shortening. In summary, TERRA RNA-DNA hybrids regulate telomere dysfunction-induced senescence in both pre- and post-crisis cells.

molecular biology↗

bioRxiv2022

A phylogenomically informed five-order system for the closest relatives of land plants

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta) [1]. There is convincing evidence that the unicellular/filamentous Zygnematophyceae--and not the morphologically more elaborate Coleochaetophyceae or Charophyceae--are the closest algal relatives of land plants [2, 3, 4, 5, 6]. Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved [7, 8]. There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago [9], but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580, and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species, and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae, and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.

evolutionary biology↗

bioRxiv2022

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs decrease. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/479353v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5712f9org.highwire.dtl.DTLVardef@9eb133org.highwire.dtl.DTLVardef@83b7b2org.highwire.dtl.DTLVardef@1912e5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗