Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger ...RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we show that Robo3.2, a receptor for the Slit family of guidance cues, is synthesized locally within axons of commissural neurons. Robo3.2 translation is induced by floor-plate-derived signals as axons cross the spinal cord midline. Robo3.2 is also a predicted target of the nonsense-mediated mRNA decay (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding.
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•Robo3.2 is locally synthesized in growth cones of commissural axons•NMD proteins are enriched in growth cones of diverse types of neurons•NMD regulates the amount of Robo3.2 synthesized in growth cones•Defects in NMD lead to abnormal axonal trajectories in spinal cord
In growth cones of spinal-cord commissural neurons, the levels of the axon guidance receptor Robo3.2 are regulated by nonsense-mediated mRNA decay (NMD), with defects in NMD leading to abnormal axonal trajectories.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The explosion in sequencing technologies has provided us with an instrument to describe mammalian transcriptomes at unprecedented depths. This has revealed that alternative splicing is used ...extensively not only to generate protein diversity, but also as a means to regulate gene expression post‐transcriptionally. Intron retention (IR) is overwhelmingly perceived as an aberrant splicing event with little or no functional consequence. However, recent work has now shown that IR is used to regulate a specific differentiation event within the haematopoietic system by coupling it to nonsense‐mediated mRNA decay (NMD). Here, we highlight how IR and, more broadly, alternative splicing coupled to NMD (AS‐NMD) can be used to regulate gene expression and how this is deregulated in disease. We suggest that the importance of AS‐NMD is not restricted to the haematopoietic system but that it plays a prominent role in other normal and aberrant biological settings.
The excision of introns may be affected by different mechanisms, which all lead to the retention of an intron in the final mRNA. As demonstrated in recent reports, intron retention is employed not only to regulate gene expression output but is also found to be deregulated in disease.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
During transcription, most eukaryotic genes generate multiple alternative cleavage and polyadenylation (APA) sites, leading to the production of transcript isoforms with variable lengths in the 3' ...untranslated region (3'UTR). In contrast to somatic cells, male germ cells, especially pachytene spermatocytes and round spermatids, express a distinct reservoir of mRNAs with shorter 3'UTRs that are essential for spermatogenesis and male fertility. However, the mechanisms underlying the enrichment of shorter 3'UTR transcripts in the developing male germ cells remain unknown. Here, we report that UPF2-mediated nonsense-mediated mRNA decay (NMD) plays an essential role in male germ cells by eliminating ubiquitous genes-derived, longer 3'UTR transcripts, and that this role is independent of its canonical role in degrading "premature termination codon" (PTC)-containing transcripts in somatic cell lineages. This report provides physiological evidence supporting a noncanonical role of the NMD pathway in achieving global 3'UTR shortening in the male germ cells during spermatogenesis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here ...we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.
Research on human and murine haematopoiesis has resulted in a vast number of gene-expression data sets that can potentially answer questions regarding normal and aberrant blood formation. To ...researchers and clinicians with limited bioinformatics experience, these data have remained available, yet largely inaccessible. Current databases provide information about gene-expression but fail to answer key questions regarding co-regulation, genetic programs or effect on patient survival. To address these shortcomings, we present BloodSpot (www.bloodspot.eu), which includes and greatly extends our previously released database HemaExplorer, a database of gene expression profiles from FACS sorted healthy and malignant haematopoietic cells. A revised interactive interface simultaneously provides a plot of gene expression along with a Kaplan-Meier analysis and a hierarchical tree depicting the relationship between different cell types in the database. The database now includes 23 high-quality curated data sets relevant to normal and malignant blood formation and, in addition, we have assembled and built a unique integrated data set, BloodPool. Bloodpool contains more than 2000 samples assembled from six independent studies on acute myeloid leukemia. Furthermore, we have devised a robust sample integration procedure that allows for sensitive comparison of user-supplied patient samples in a well-defined haematopoietic cellular space.
Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein ...lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16
. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.
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IJS, NUK, SBMB, UL, UM, UPUK
DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated ...in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.
Summary
Recent advances in sequencing technologies have allowed for the identification of recurrent mutations in acute myeloid leukaemia (AML). The transcription factor CCAAT enhancer binding protein ...alpha (CEBPA) is frequently mutated in AML, and biallelic CEBPA‐mutant AML was recognised as a separate disease entity in the recent World Health Organization classification. However, CEBPA mutations are co‐occurring with other aberrations in AML, and together these lesions form the clonal hierarchy that comprises the leukaemia in the patient. Here, we aim to review the current understanding of co‐occurring mutations in CEBPA‐mutated AML and their implications for disease biology and clinical outcome. We will put emphasis on patterns of cooperation, how these lesions cooperate with CEBPA mutations and the underlying potential molecular mechanisms. Finally, we will relate this to patient outcome and future options for personalised medicine.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with ...population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.