Biology subjects

Heyd, F.

Publications and source records attributed to Heyd, F..

11 recordsLinked to original sources

bioRxiv2024

AFC kinases function as thermosensors that regulate warm temperature-responsive growth in Arabidopsis

Plants respond to elevated temperatures with enhanced elongation growth that depends on rapid transcriptional, post-transcriptional, and post-translational reprogramming. However, it is unclear how temperature information integrates with the splicing machinery to establish warm temperature-dependent splicing patterns. In animals, CDC2-LIKE KINASES (CLKs) function as body temperature sensors that control temperature-dependent splicing via phosphorylation of serine/arginine-rich (SR) proteins. Here we demonstrate that the CLK-homologous ARABIDOPSIS FUS3-COMPLEMENTING (AFC) kinases likewise regulate post-transcriptional RNA processing to control warm temperature-dependent growth in Arabidopsis. The contrasting temperature-activity profiles of the three AFCs depend on specific structural elements, including a conserved activation segment within the kinase domain. Combining protein structure prediction with site-directed mutagenesis, we provide insights into structural features that determine the different temperature-activity profiles of the three AFC paralogs. Analyses of afc mutant plants demonstrate their role in establishing temperature-dependent splicing patterns and thermomorphogenic hypocotyl elongation. Finally, our data indicate SR34 and SR34a as phosphorylation targets mediating temperature-dependent hypocotyl elongation downstream of AFCs. In conclusion, our study provides evidence that temperature-controlled AFC activity is evolutionarily conserved between plants and animals and implicates AFCs in the control of thermomorphogenesis.

plant biology↗

bioRxiv2024

Comparison of the CDC2-like kinase family across eukaryotes highlights the functional conservation of these unique biological thermometers

The family of CDC2-like kinases (CLKs) play a crucial role in regulating alternative splicing (AS), a process fundamental to eukaryotic gene expression and adaptation. Of particular interest, these enzymes exhibit unique responsiveness to minor temperature shifts, enabling them to modulate AS accordingly. Dysregulated CLK expression is linked to a wide variety of human diseases, establishing them as promising therapeutic targets. Despite the importance of CLKs, limited research has explored the genetic and functional diversification of this gene family. This report investigates the evolutionary origins, diversification, and functional implications of CLKs across major eukaryotic lineages through phylogenetic and structural comparisons. Our data demonstrate these kinases are prevalent throughout eukaryotes, with the original gene (which shares orthology to human CLK2), dating back to the Last Eukaryotic Common Ancestor. We identified three key duplication events in vertebrates, highlighting how this gene family has expanded and diversified in complex metazoans. Despite two instances of CLK paralog loss in vertebrate lineages, CLKs remain prevalent throughout metazoans, suggesting they are essential for complex eukaryotic life. Structural comparisons across diverse eukaryotes demonstrate kinase domain conservation, which is in line with their maintained function in AS regulation. While their N-terminal regions vary significantly in amino acid sequence, the function of this domain to regulate phosphorylation of AS factors is conserved, albeit in a species-specific manner. CLKs exhibit unique thermo-sensitive properties across diverse species, challenging conventional enzymatic behaviour. This temperature regulation, mediated by their kinase activation segment, is characterised by increased activity at lower physiological temperatures. The conservation of this structure, and a thermo-sensitive amino acid motif within it, suggests this was an ancient adaptation for responding to environmental cues. Species-specific temperature profiles highlight the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions including extreme temperatures. Our analysis expands the understanding of CLK biology across diverse eukaryotes and connects insights from model organisms to human biology.

evolutionary biology↗

bioRxiv2024

Stabilizing mammalian RNA thermometer confers neuroprotection in subarachnoid hemorrhage

Mammals tightly regulate their core body temperature, yet how cells sense and respond to small temperature changes at the molecular level remains incompletely understood. Here, we discover a significant enrichment of RNA G-quadruplex (rG4) motifs around splice sites of cold-repressed exons. These thermosensing RNA structures, when stabilized, mask splice sites, reducing exon inclusion. Focusing on cold-induced neuroprotective RBM3, we demonstrate that rG4s near splice sites of a cold-repressed poison exon are stabilized at low temperatures, leading to exon exclusion. This enables evasion of nonsense-mediated decay, increasing RBM3 expression at cold. Additionally, increasing intracellular potassium concentration stabilizes rG4s and enhances RBM3 expression, leading to RBM3-dependent neuroprotection in a mouse model of subarachnoid hemorrhage. Our findings unveil a mechanism how mammalian RNAs directly sense temperature and potassium perturbations, integrating them into gene expression programs. This opens new avenues for treating diseases arising from splicing defects and disorders benefiting from therapeutic hypothermia. HighlightsO_LIrG4s are enriched near splice sites of cassette exons repressed upon cold shock C_LIO_LIrG4s act as RNA thermometers in mammals by controlling accessibility of splice sites C_LIO_LIrG4 stability mediates temperature-dependent RBM3 expression ex vivo and in vivo C_LIO_LIStabilizing rG4s in RBM3 exon 3a reduces brain damage in subarachnoid hemorrhage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/594628v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@e1d625org.highwire.dtl.DTLVardef@b22d21org.highwire.dtl.DTLVardef@1b0faeaorg.highwire.dtl.DTLVardef@294ac1_HPS_FORMAT_FIGEXP M_FIG C_FIG Here is a refined model of RNA G-quadruplexes acting as evolutionarily conserved thermo- and potassium sensors, modulating alternative splicing in mammals. RNA G-quadruplexes (rG4s) function as reversible temperature sensors, impacting alternative splicing dynamics. In low temperatures or high potassium conditions, rG4s can mask surrounding splice sites, rendering these sites inaccessible, thereby promoting exon skipping. Conversely, at high temperatures or under low potassium conditions, rG4s become destabilized, allowing splice sites to be exposed, and facilitating efficient exon inclusion. RBM3, a well-known cold-induced protein with neuroprotective functions, harbors a poison exon with rG4s around splice sites, that, upon inclusion, triggers NMD (non-sense mediated decay) of the RBM3 mRNA. Under low temperatures or high potassium conditions, rG4s shield the splice sites, leading to poison exon skipping and increased RBM3 expression. Stabilization of these rG4s through increased K+ promotes poison exon skipping, enabling escape from NMD, and ultimately elevating RBM3 expression. Notably, 4-AP, a clinically used pan voltage-gated potassium channel blocker, protects against neuronal damage in a subarachnoid hemorrhage mouse model in an RBM-dependent manner. (ISS: intronic splicing silencer, ESE: exon splicing enhancer, ACA: anterior cerebral artery, MCA: middle cerebral artery, PPA: pterygopalatine artery, ICA: internal carotid artery, ECA: external carotid artery, CCA: common carotid artery)

molecular biology↗

bioRxiv2024

Septin-associated PIPKIγ splice variants drive centralspindlin association with the midbody via PI(4,5)P2

Mammalian cytokinesis critically depends on the phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] which serves as docking site for crucial components of the cytokinetic machinery at the plasma membrane. PI(4,5)P2 supports several stages of cytokinesis, including actomyosin ring assembly and constriction, membrane tethering of spindle microtubules, and midbody organization. How these various activities of PI(4,5)P2 and the underlying mechanisms of local PI(4,5)P2 synthesis are orchestrated in space and time has remained elusive. Here, we identify a pivotal role of septin-binding splice variants of PIPKI{gamma} that couple nanoscale PI(4,5)P2 synthesis at the ingressing cleavage furrow to late midbody formation. Depletion of PIPKI{gamma} isoforms causes multinucleation and perturbs anillin and septin deposition at the intercellular bridge and at the midbody. These defects are rescued by wild-type kinase, but not by septin binding-deficient or catalytically inactive PIPKI{gamma} variants. We further show that both, septins and PIPKI{gamma} form a complex with centralspindlin, and thereby facilitate the recruitment of centralspindlin to the midbody. Taken together, our findings establish septin-associated PIPKI{gamma} isoforms as key regulators of midbody organization that act through generating a local pool of PI4,5P2 required for centralspindlin recruitment and maintenance at the midbody, and for septin association with microtubules.

cell biology↗

bioRxiv2023

Identification of a link between splicing and endoplasmic reticulum proteostasis

The role of general splicing in endoplasmic reticulum (ER)-proteostasis remains poorly understood. Here, we identify SNRPB, a component of the spliceosome, as a novel regulator of export from the ER. Mechanistically, SNRPB regulates the splicing of components of the ER export machinery, including Sec16A, a regulator of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in intracellular accumulation of type-1 collagen as well as bone defects reminiscent of CCMS. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This indicates that the role of SNRPB in osteogenesis is linked to its effects on ER export. Finally, we show that SNRPB is a target for the unfolded protein response (UPR), which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights SNRPB as a novel node in the proteostasis network, shedding light on CCMS pathophysiology.

cell biology↗

bioRxiv2022

ASO targeting temperature-controlled RBM3 poison exon splicing prevents neurodegeneration in vivo

Neurodegenerative diseases are increasingly prevalent in the aging population, yet currently no disease-modifying treatments are available. Increasing the expression of the cold-shock protein, RBM3, through therapeutic hypothermia is remarkably neuroprotective, but cooling poses a health risk itself, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for cold-induced RBM3 expression. Genetic removal or ASO-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increase of RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of prion protein. RBM3-inducing ASOs could thus broadly deliver protection in humans in conditions ranging from acute brain injury to Alzheimers disease. One sentence summaryInducing cold shock protein RBM3 by modulating its alternative splicing at normothermia is neuroprotective in vivo

molecular biology↗

bioRxiv2022

Branch point evolution controls species-specific alternative splicing and regulates long term potentiation

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase II{beta} (CaMKII{beta}) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific Camk2{beta} alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing a paradigm for cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9 we introduced a weaker human branch point into the mouse genome, resulting in human-like CAMK2B splicing in the brain of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled, species-specific alternative splicing with a fundamental function in learning and memory.

molecular biology↗

bioRxiv2021

Targeting the MYC interaction network in B-cell lymphoma via histone deacetylase 6 inhibition

Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.

cancer biology↗

bioRxiv2020

Alternative splicing coupled nonsense-mediated decay shapes the temperature-dependent transcriptome

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature-sensitive thermometer-like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate mRNA variants containing premature termination codons, which are degraded by nonsense-mediated decay (NMD). Here we show extensive coupling of body temperature-controlled AS to NMD, leading to global control of temperature-dependent gene expression (GE). Temperature-controlled NMD-inducing splicing events are evolutionarily conserved and pervasively found within RNA-binding proteins, including most SR proteins. NMD-inducing exons are essential for rhythmic GE of SR proteins and have a global role in establishing temperature-dependent rhythmic GE profiles, both, in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature-driven AS-NMD as an evolutionary ancient, core clock-independent mechanism to generate rhythmic GE.

molecular biology↗

bioRxiv2019

A homozygous genome-edited Sept2-EGFP fibroblast cell line

Septins are a conserved, essential family of GTPases that interact with actin, microtubules and membranes and form scaffolds and diffusion barriers in cells. Several of the 13 known mammalian septins assemble into nonpolar, multimeric complexes that can further polymerize into filamentous structures. While some GFP-coupled septins have been described, overexpression of GFP-tagged septins often leads to artifacts in localization and function. To overcome this ubiquitous problem, we have here generated a genome-edited rat fibroblast cell line expressing Septin 2 (Sept2) coupled to enhanced green fluorescent protein (EGFP) from both chromosomal loci.\n\nWe characterize these cells by genomic polymerase chain reaction (PCR) for genomic integration, by western blot and reverse transcriptase-PCR for expression, by immunofluorescence and immunoprecipitation for the colocalization of septins with one another and cellular structures and for complex formation of different septins. By live cell imaging, proliferation and migration assays we investigate proper function of septins in these cells.\n\nWe find that EGFP is incorporated into both chromosomal loci and only EGFP-coupled Sept2 is expressed in homozygous cells. We find that endogenous Sept2-EGFP exhibits expression levels, localization and incorporation into cellular septin complexes similar to the wt in these cells. The expression level of other septins is not perturbed and cell division and cell migration proceed normally. We expect our cell line to be a useful tool for the cell biology of septins, especially for quantitative biology.

cell biology↗

bioRxiv2018

Genome-wide identification of alternative splicing events that regulate protein transport across the secretory pathway

Alternative splicing (AS) strongly increases proteome diversity and functionality in eukaryotic cells. Protein secretion is a tightly-controlled process, especially in a tissue-specific and differentiation-dependent manner. While previous work has focussed on transcriptional and posttranslational regulatory mechanisms, the impact of AS on the secretory pathway remains largely unexplored. Here we integrate a published screen for modulators of protein transport and RNA-Seq analyses to identify over 200 AS events as secretion regulators. We confirm that splicing events along all stages of the secretory pathway regulate the efficiency of protein transport using Morpholinos and CRISPR/Cas9. We furthermore show that these events are highly tissue-specific and adapt the secretory pathway during T-cell activation and adipocyte differentiation. Our data substantially advance the understanding of AS functionality, add a new regulatory layer to a fundamental cell biological process and provide a resource of alternative isoforms that control the secretory pathway.

cell biology↗