Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is ...based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.
Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation, or produce ...truncated proteins that differ in localization or function. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5′-transcribed regions. To discern how Set3C affects gene ...expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.
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► Set3C negatively regulates the kinetics of transcription induction at some genes ► Set3C inhibits activity of cryptic internal promoters in 5′-transcribed regions ► Overlapping ncRNA transcription localizes H3K4me2 to Set3C-repressed promoters ► ncRNA transcription and Set3C cooperate to fine-tune the kinetics of gene induction
The Set3 histone deacetylase complex targets the promoters of yeast genes that are overlapped by noncoding transcripts, thus modulating their response to changing growth conditions. The ncRNAs recruit Set3 by promoting the deposition of histone methylation marks that are recognized by the deacetylase complex.
Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we ...developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.
RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and ...comprehensive approach, termed “interactome capture,” to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
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► Interactome capture identifies hundreds of RBPs previously unknown to bind RNA ► Some globular and disordered regions represent unorthodox binding architectures ► Many RNA binders are linked to Mendelian disease ► Metabolic enzymes moonlighting as RBPs
Interactome capture identifies the landscape of mRNA-binding proteins in mammalian cells, revealing new roles for metabolic enzymes and a propensity for globular and unstructured regions to associate with messages.
The bone marrow constitutes the primary site for life-long blood production and skeletal regeneration. However, its cellular and spatial organization remains controversial. Here, we combine ...single-cell and spatially resolved transcriptomics to systematically map the molecular, cellular and spatial composition of distinct bone marrow niches. This allowed us to transcriptionally profile all major bone-marrow-resident cell types, determine their localization and clarify sources of pro-haematopoietic factors. Our data demonstrate that Cxcl12-abundant-reticular (CAR) cell subsets (Adipo-CAR and Osteo-CAR) differentially localize to sinusoidal and arteriolar surfaces, act locally as 'professional cytokine-secreting cells' and thereby establish peri-vascular micro-niches. Importantly, the three-dimensional bone-marrow organization can be accurately inferred from single-cell transcriptome data using the RNA-Magnet algorithm described here. Together, our study reveals the cellular and spatial organization of bone marrow niches and offers a systematic approach to dissect the complex organization of whole organs.
The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two ...catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.
► Genome-wide analyses reveal specific function of the exosome catalytic subunits ► Most intron-containing pre-mRNAs are degraded in splicing-proficient cells ► Large amounts of tRNA precursors are degraded by the exosome in wild-type cells ► Kinetic competition with degradation promotes quality control of RNA processing
Combining the precise parallaxes and optical photometry delivered by Gaia’s second data release with the photometric catalogues of Pan-STARRS1, 2MASS, and AllWISE, we derived Bayesian stellar ...parameters, distances, and extinctions for 265 million of the 285 million objects brighter than G = 18. Because of the wide wavelength range used, our results substantially improve the accuracy and precision of previous extinction and effective temperature estimates. After cleaning our results for both unreliable input and output data, we retain 137 million stars, for which we achieve a median precision of 5% in distance, 0.20 mag in V-band extinction, and 245 K in effective temperature for G ≤ 14, degrading towards fainter magnitudes (12%, 0.20 mag, and 245 K at G = 16; 16%, 0.23 mag, and 260 K at G = 17, respectively). We find a very good agreement with the asteroseismic surface gravities and distances of 7000 stars in the Kepler, K2-C3, and K2-C6 fields, with stellar parameters from the APOGEE survey, and with distances to star clusters. Our results are available through the ADQL query interface of the Gaia mirror at the Leibniz-Institut für Astrophysik Potsdam (gaia.aip.de) and as binary tables at data.aip.de. As a first application, we provide distance- and extinction-corrected colour-magnitude diagrams, extinction maps as a function of distance, and extensive density maps. These demonstrate the potential of our value-added dataset for mapping the three-dimensional structure of our Galaxy. In particular, we see a clear manifestation of the Galactic bar in the stellar density distributions, an observation that can almost be considered direct imaging of the Galactic bar.
Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such ...stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.
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•Unipotent SL-MkPs are maintained in a quiescent, but primed, state during homeostasis•Inflammation instructs post-transcriptional Mk protein synthesis in SL-MkPs•Inflammation drives efficient cell cycle activation and maturation of SL-MkPs•Activation of SL-MkPs rapidly replenishes the platelet pool during acute inflammation
Haas et al. show that inflammatory signaling activates post-transcriptional protein synthesis, maturation, and cell cycle induction in quiescent, but primed, stem-like megakaryocyte progenitors. This emergency program efficiently prevents otherwise life-threatening platelet depletion during acute inflammation.
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