Biology subjects

Rieger, M. A.

Publications and source records attributed to Rieger, M. A..

12 recordsLinked to original sources

bioRxiv2026

Single-Cell Mapping of tRNA Expression Dynamics Across Human Hematopoiesis

How transfer RNA (tRNA) expression changes as hematopoietic stem cells are specified into human blood cell types remains unknown. tRNAs translate the 64-codon genetic code into the 21-amino acid protein code, and their relative abundance can influence proteomic output. Here, we introduce simultaneous single-cell tRNA and mRNA sequencing (sc-STM-seq), a scalable, high-throughput approach for profiling tRNAs alongside mRNA transcriptomes in individual cells. Applying sc-STM-seq to human bone marrow, we map tRNA expression and splicing across hematopoietic differentiation trajectories and identify tRNAs associated with stemness, differentiation, and specific cell lineages. Our study provides an atlas of the hematopoietic tRNA landscape and establishes tRNA expression as a previously underappreciated layer of cellular heterogeneity during human blood cell development.

cell biology↗

bioRxiv2026

Variation of Human Transfer RNA Demand and Supply

Codons function as translation units in open-reading-frames (ORF) of genes to encode for proteins. Transfer RNAs (tRNAs) mediate the connection of every codon to its cognate amino acid. Despite the cooperation between messenger and transfer RNA during translation, approaches to integrate codon usage and tRNA quantities remain to be established. Using matched mRNA- and tRNA-sequencing of peripheral blood cells, we apply a precision-biology approach quantitatively integrating transcriptomic codon- and corresponding tRNA-abundance. Thereby, we classify codons as highly or lowly supplied and compare optimality of synonymous codons. Additionally, we describe substantial differences regarding the conservation of a codons tRNA-supply among healthy donors. A meta-ORF-analysis demonstrates depletion of lowly supplied codons at translation start sites. Discrepancy between codon- and tRNA-abundance, and codon-preference depending on the distance to the translation start site, seem to be non-random and could affect translational speed and thus provide a novel level of regulation of protein abundance.

systems biology↗

bioRxiv2025

Endothelial expression of ZBTB16 protects against cardiac aging

Background and aimAging significantly increases the risk of cardiovascular diseases, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms mediating EC-dependent cardiac aging and identifies a critical role of Zinc finger and BTB domain-containing protein 16 (ZBTB16). MethodsChromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses were performed on aged hearts to identify age-related regulatory changes. Functional studies using genetic models, assessed cardiac aging phenotypes. In vitro assays examined EC senescence and secretory profiles, while co-culture experiments analyzed the impact of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested the potential for ZBTB16 to mitigate aging-associated dysfunction. ResultsAged hearts exhibited decreased chromatin accessibility and expression of the transcription factor ZBTB16 in both human and mice. Loss of ZBTB16 in young mice, including Zbtb16 haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. Supernatants from ZBTB16-deficient ECs activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified key downstream targets of ZBTB16, including nuclear receptor-interacting protein 1 (NRIP1). ZBTB16 suppressed NRIP1 expression, limiting fibroblast activation and pro-fibrotic signaling. ConclusionsZBTB16 is a key regulator of endothelial function, maintaining vascular niche homeostasis and mitigating aging-associated cardiac dysfunction. Its loss promotes EC senescence and pro-fibrotic signaling, contributing to diastolic dysfunction. Overexpression of ZBTB16 presents a potential therapeutic strategy for preserving cardiac function during aging. These findings establish a novel role for ZBTB16 in endothelial aging and cardiovascular disease prevention. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/681100v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a56e2org.highwire.dtl.DTLVardef@13e23fforg.highwire.dtl.DTLVardef@ad681eorg.highwire.dtl.DTLVardef@8eca8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Differentiation hierarchy in adult B cell acute lymphoblastic leukemia at clonal resolution

While a differentiation hierarchy with leukemia-initiating stem cells (LICs) at the apex is well documented for acute myeloid leukemia, the existence of LICs and their trajectories in B cell acute lymphoblastic leukemia (B-ALL) are debated. B-ALL is a malignant disease displaying considerable phenotypical and functional heterogeneity, yet the underlying cellular organization remains largely elusive. This study aims to investigate the hierarchical landscape of B-ALL by combining barcoding and multiome single cell data to unveil the differentiation architecture and temporal dynamics of leukemic differentiation at clonal and single cell resolution in vivo. Single-cell transcriptome and AbSeq analysis of lentiviral barcoded and transplanted patient-derived B-ALL cells revealed interconnected subpopulations, which could be prospectively isolated via surface markers and exhibited distinct leukemogenicity. Barcode tracking demonstrated the ability of cells to differentiate from a source cluster into differentiated progeny. Using machine learning we identified expression patterns predicting their differentiation potential of each barcoded cell within the immature cell compartment. To determine the dynamical properties of clones, we have designed a mathematical model that simulates the development of these subpopulations from a clonally diverse stem cell compartment. The model confirmed the most likely cluster of origin, supporting our functional data, and revealed the variability in dynamical properties between clones, while largely excluding plasticity. Hence, our work demonstrates a unidirectional differentiation in B-ALL with an immature population exhibiting a high leukemogenic potential. Key pointsO_LIA differentiation hierarchy with distinct leukemogenesis potential in B-ALL is demonstrated via clonal tracing and mathematical modeling. C_LIO_LIClonal differentiation behavior, while heterogeneous, is cell intrinsically controlled and predictable, yet independent from expansion. C_LI

cancer biology↗

bioRxiv2024

Inhibition of IAPs induces programmed cell death and inflammatory signaling in patient-derived metastatic breast cancer organoids

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects therapy success and remains a major challenge. Representative and translational models to understand, manipulate and cultivate advanced BC and to model PCD resistance are therefore urgently required. Smac mimetics are promising compounds to circumvent apoptosis resistance and are able to induce caspase-independent necroptosis, a lytic and inflammatory mode of PCD. Here, we apply primary, patient-derived human mammary organoids (hMOs) to investigate alternative forms of PCD to overcome apoptosis resistance. Using time lapse brightfield with immunofluorescent confocal microscopy, biochemistry and gene expression analysis, we demonstrate that Smac mimetics induce apoptosis in primary hMOs. By mimicking apoptosis resistance via caspase inhibition, hMOs undergo necroptosis, associated with expression and secretion of inflammatory mediators. Inhibition of linear ubiquitination by the LUBAC inhibitor HOIPIN-8 prevents necroptosis, as well as the expression and release of inflammatory mediators in hMOs. Our findings demonstrate that primary hMOs are effective models to model, study and manipulate PCD responses and inflammation in in primary BC organoids and open new therapeutic screening options for chemotherapy-resistant BC.

cancer biology↗

bioRxiv2021

The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

Methamphetamine addiction remains a major public health concern in the United States that has paralleled the opioid epidemic. Psychostimulant use disorders have a heritable genetic component that remains unexplained. Methamphetamine targets membrane and vesicular transporters to increase synaptic dopamine, norepinephrine, and serotonin. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying methamphetamine behavioral sensitivity. Hnrnph1 encodes the RNA-binding protein hnRNP H1 that is ubiquitously expressed in neurons throughout the brain. Gene-edited mice with a heterozygous frameshift deletion in Hnrnph1s first coding exon of showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H with methamphetamine neurobehavioral effects, we surveyed the mRNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine in wild-type male and female C57BL/6J mice. Methamphetamine induced changes in RNA-binding targets of hnRNP H in mice, including differential binding to 3UTR targets and multiple enriched mRNAs involved in synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on Cacna2d2 as a suggestive target, with differences in hnRNP H binding, gene expression and splicing following methamphetamine treatment (2 mg/kg, i.p.). Furthermore, pre-treatment with pregabalin, an inhibitor of 2{delta}2 and 2{delta}1 voltage-gated calcium channel subunits, attenuated methamphetamine-induced locomotor activity in male and female mice, supporting a role for Cacna2d1/d2 in methamphetamine locomotor stimulant sensitivity. Our study identifies a dynamic hnRNP H RNA targetome that can rapidly and adaptively respond to methamphetamine to regulate gene expression and likely synaptic plasticity and behavior.

neuroscience↗

bioRxiv2021

A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with genomic elements containing variants from ASD patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This new technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.

genetics↗

bioRxiv2020

Hematopoietic stem cells fail to regenerate following inflammatory challenge.

Hematopoietic stem cells (HSCs) are canonically defined by their capacity to maintain the HSC pool via self-renewal divisions. However, accumulating evidence suggests that HSC function is instead preserved by sustaining long-term quiescence. Here, we study the kinetics of HSC recovery in mice, following an inflammatory challenge that induces HSCs to exit dormancy. Repeated inflammatory challenge resulted in a progressive depletion of functional HSCs, with no sign of later recovery. Underlying this observation, label retention experiments demonstrated that self-renewal divisions were absent or extremely rare during challenge, as well as during any subsequent recovery period. While depletion of functional HSCs held no immediate consequences, young mice exposed to inflammatory challenge developed blood and bone marrow hypocellularity in old age, similar to elderly humans. The progressive, irreversible attrition of HSC function demonstrates that discreet instances of inflammatory stress can have an irreversible and therefore cumulative impact on HSC function, even when separated by several months. These findings have important implications for our understanding of the role of inflammation as a mediator of dysfunctional tissue maintenance and regeneration during ageing.

cell biology↗

bioRxiv2020

CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses.

Quaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3 UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocytes territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

neuroscience↗

bioRxiv2018

CLIP-Seq and massively parallel functional analysis of the CELF6 RNA binding protein reveals a role in destabilizing synaptic gene mRNAs through interaction with 3’UTR elements in vivo

CELF6 is a RNA-binding protein in a family of proteins with roles in human health and disease, however little is known about the mRNA targets or in vivo function of this protein. We utilized CLIP-Seq to identify, for the first time, in vivo targets of CELF6 and identify hundreds of transcripts bound by CELF6 in the brain. We found these are disproportionately mRNAs coding for synaptic proteins. We then conducted functional validation of these targets, testing greater than 400 CELF6 bound sequence elements for their activity, applying a massively parallel reporter assay framework to evaluation of the CLIP data. We also mutated potential binding motifs within these elements and tested their impact. This comprehensive analysis led us to ascribe a previously unknown function to CELF6: we found bound elements were generally repressive of translation, that CELF6 further enhances this repression via decreasing RNA abundance, and this process was dependent on UGU-rich sequence motifs. This greatly extends the known role for CELF6, which had previously been defined only as a splicing factor. We further extend these findings by demonstrating the same function for CELF3, CELF4, and CELF5. Finally, we demonstrate that the CELF6 targets are derepressed in CELF6 mutant mice in vivo, confirming this new role in the brain. Thus, our study demonstrates that CELF6 and other sub-family members are repressive CNS RNA-binding proteins, and CELF6 downregulates specific mRNAs in vivo.

molecular biology↗

bioRxiv2018

Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia

Recent studies implicated that long non-coding RNAs (lncRNAs) may play a role in the progression and development of acute lymphoblastic leukemia, however, this role is not yet clear. In order to unravel the role of lncRNAs associated with B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) subtypes, we performed transcriptome sequencing and DNA methylation array across 82 BCP-ALL samples from three molecular subtypes (DUX4, Ph-like, and Near Haploid or High Hyperdiploidy). Unsupervised clustering of BCP-ALL samples on the basis of their lncRNAs on transcriptome and DNA methylation profiles revealed robust clusters separating three molecular subtypes. Using extensive computational analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific lncRNAs with altered expression and methylation patterns from three subtypes of BCP-ALL. By analyzing the co-expression of subtype-specific lncRNAs and protein-coding genes, we inferred key molecular processes in BCP-ALL subtypes. A strong correlation was identified between the DUX4 specific lncRNAs and activation of TGF-{beta} and Hippo signaling pathways. Similarly, Ph-like specific lncRNAs were correlated with genes involved in activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific differentially expressed lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we showed a set of epigenetically altered lncRNAs facilitating the expression of tumor genes located at their cis location. Overall, our study provides a comprehensive set of novel subtype and relapse-specific lncRNAs in BCP-ALL. Our findings suggest a wide range of molecular pathways are associated with lncRNAs in BCP-ALL subtypes and provide a foundation for functional investigations that could lead to new therapeutic approaches.\n\nAuthor SummaryAcute lymphoblastic leukemia is a heterogeneous blood cancer, with multiple molecular subtypes, and with high relapse rate. We are far from the complete understanding of the rationale behind these subtypes and high relapse rate. Long non-coding (lncRNAs) has emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. LncRNAs does not code for proteins and represent around 70% of human transcripts. Recently, there are a number of studies used lncRNAs expression profile in the classification of various cancers subtypes and displayed their correlation with genomic, epigenetic, pathological and clinical features in diverse cancers. Therefore, lncRNAs can account for heterogeneity and has independent prognostic value in various cancer subtypes. However, lncRNAs defining the molecular subtypes of BCP-ALL are not portrayed yet. Here, we describe a set of relapse and subtype-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining how lncRNAs are involved in the pathogenesis of disease and the relevance in the stratification of BCP-ALL subtypes.

cancer biology↗

bioRxiv2017

Maternal SSRI treatment during offspring development results in long-term behavioral, cellular, and neuroimaging disruptions

Serotonergic dysregulation is implicated in psychiatric disorders, including autism spectrum disorders (ASD). Epidemiological studies suggest selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy may increase ASD risk in offspring, however it is unclear from these studies whether ASD susceptibility is related to the maternal diagnosis or if treatment poses additional risk. Here, we exposed mouse dams to fluoxetine and characterized the offspring to isolate possible effects of SSRI exposure on ASD-relevant behaviors. We demonstrate social communication and interaction deficits and repetitive behaviors, with corresponding dendritic morphology changes in pertinent brain regions. Also, using a novel application of optical intrinsic signal imaging, we show altered stimulus-evoked cortical response and region-specific decreases in functional connectivity. These findings indicate drug exposure alone is sufficient to induce long-term behavioral, cellular, and hemodynamic-response disruptions in offspring, thus contributing to our understanding of ASD pathogenesis, risk and mechanism, as well as the developmental role of serotonin.

neuroscience↗