استكشف المجموعة الواسعة من العناوين المتاحة.

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single‐cell transcriptome analysis of tumors and matched non‐cancerous tissues of twelve colorectal cancer patients. We defined patient‐overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen‐activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient‐derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen‐activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR‐BRAF‐MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non‐genetic cancer cell heterogeneity and re‐routing of trajectories as a response to targeted therapy. SYNOPSIS Colorectal cancer (CRC) cells can adopt a range of transcriptomic states. This study uses single cell RNA sequencing of primary CRC tissue and organoids to identify patient‐overarching CRC cell transcriptome clusters. RNA metabolic labelling indicates preferred CRC cell developmental trajectories. CRC cells of multiple patients clustered into six groups – termed TC1‐4, Goblet‐like, and stem‐like – characterized by differential transcriptional footprints of oncogenic signaling pathways. CRC organoid cells develop along a decreasing MAPK gradient. Experimental targeting of EGFR‐MAPK in CRC organoids re‐routes developmental trajectories. Clinically relevant inhibition of EGFR‐MAPK can result in preferential CRC cell development towards endpoints expressing high levels of stem cell markers. Graphical Abstract Colorectal cancer (CRC) cells can adopt a range of transcriptomic states. This study uses single cell RNA sequencing of primary CRC tissue and organoids to identify patient‐overarching CRC cell transcriptome clusters. RNA metabolic labelling indicates preferred CRC cell developmental trajectories.

Tissue dissociation, a crucial step in single‐cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell‐type‐specific dissociation response programs in zebrafish larvae, and we observed sample‐to‐sample variation in the dissociation response of mouse cardiomyocytes despite well‐controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response. Synopsis A new approach shows that RNA labelling can be used to measure the cellular response to tissue dissociation, a major confounding factor in single‐cell transcriptomics. The dissociation response is partially cell‐type specific. Single‐cell RNA labelling allows measuring the cellular dissociation response, a major confounding factor in single‐cell transcriptomics. The dissociation response is comprised of a core signature that is shared across tissue types and replicates as well as sample‐ and cell‐type‐specific programs. The dissociation of the mouse hippocampus can lead to the activation of microglia. Graphical Abstract A new approach shows that RNA labelling can be used to measure the cellular response to tissue dissociation, a major confounding factor in single‐cell transcriptomics. The dissociation response is partially cell‐type specific.

mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells. Proteins are made by joining together building blocks called amino acids into strings. The proteins are ‘translated’ from genetic sequences called mRNA molecules. These sequences can be thought of as series of ‘letters’, which are read in groups of three known as codons. Molecules called tRNAs recognize the codons and add the matching amino acids to the end of the protein. Each tRNA can recognize one or several codons, and the levels of different tRNAs inside the cell vary. There are 61 codons that code for amino acids, but only 20 amino acids. This means that some codons produce the same amino acid. Despite this, there is evidence to suggest that not all of the codons that produce the same amino acid are exactly equivalent. In bacteria, yeast and zebrafish, some codons seem to make the mRNA molecule more stable, and others make it less stable. This might help the cell to control how many proteins it makes. It was not clear whether the same is true for humans. To find out, Wu et al. used three separate methods to examine mRNA stability in four types of human cell. Overall, the results revealed that some codons help to stabilize the mRNA, while others make the mRNA molecule break down faster. The effect seems to depend on the supply of tRNAs that have a charged amino acid; mRNA molecules were more likely to self-destruct in cells that contained codons with low levels of the tRNA molecules. Wu et al. also found that conditions in the cell can alter how strongly the codons affect mRNA stability. For example, a cell that has been infected by a virus reduces translation. Under these conditions, the identity of the codons in the mRNA has less effect on the stability of the mRNA molecule. Changes to protein production happen in many diseases. Understanding what controls these changes could help to reveal more about our fundamental biology, and what happens when it goes wrong.

Inactivation of cyclin‐dependent kinase 12 (CDK12) characterizes an aggressive sub‐group of castration‐resistant prostate cancer (CRPC). Hyper‐activation of MYC transcription factor is sufficient to confer the CRPC phenotype. Here, we show that loss of CDK12 promotes MYC activity, which renders the cells dependent on the otherwise non‐essential splicing regulatory kinase SRSF protein kinase 1 (SRPK1). High MYC expression is associated with increased levels of SRPK1 in patient samples, and overexpression of MYC sensitizes prostate cancer cells to SRPK1 inhibition using pharmacological and genetic strategies. We show that Endovion (SCO‐101), a compound currently in clinical trials against pancreatic cancer, phenocopies the effects of the well‐characterized SRPK1 inhibitor SRPIN340 on nascent transcription. This is the first study to show that Endovion is an SRPK1 inhibitor. Inhibition of SRPK1 with either of the compounds promotes transcription elongation, and transcriptionally activates the unfolded protein response. In brief, here we discover that CDK12 inactivation promotes MYC signaling in an SRPK1‐dependent manner, and show that the clinical grade compound Endovion selectively targets the cells with CDK12 inactivation. Inactivation of the transcription elongation kinase cyclin‐dependent kinase 12 (CDK12) stimulates MYC signaling. MYC serves as a transcriptional amplifier and makes cancer cells dependent on SRSF protein kinase 1 (SRPK1). We propose that inactivating mutations and deletion of the CDK12 gene are biomarkers of sensitivity to SRPK1 inhibitors.

Background Early embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately. Results By employing SLAM-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional activation events and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA-430 function, a key post transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression. Conclusion These insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. The findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions.

Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.

Background Phosphorylation and O-GlcNAcylation are the key modifications regulating RNA Polymerase II (RNA Pol II)-driven transcription. Transcriptional kinases, cyclin-dependent kinase 7 (CDK7), CDK9 and CDK12 phosphorylate RNA Pol II, whereas O-GlcNAcylation is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Currently, no study has systematically evaluated how inhibiting each of these enzyme activities impacts the assembly of the appropriate protein complexes on the polymerase and the maturation of mRNA. Methods Here, we systematically evaluate remodeling of RNA Pol II interactome and effects on the nascent mRNA maturation by using mass spectrometry and SLAM-seq, respectively. For validation, we rely predominantly on analysis of intronic polyadenylation (IPA) sites, mitochondrial flux assays (Seahorse), western blotting and patient data. Results We show that OGT / OGA inhibition reciprocally affect protein recruitment to RNA Pol II, and appropriate O-GlcNAcylation levels are required for optimal function of the RNA Pol II complex. These paradoxical effects are explained through IPA, because despite being prematurely poly-adenylated, these mRNAs are scored as mature in SLAM-seq. Unlike previously proposed, we show that, similar to inhibition of CDK12, also targeting CDK9 stimulates transcription of short genes at the cost of long genes. However, our systematic proteomic- and IPA-analysis revealed that these effects are mediated by distinct molecular mechanisms: CDK9 inhibition leads to a failure of recruiting Integrator complex to RNA Pol II, and we then show that depletion of Integrator subunits phenocopy the gene length-dependent effects. In contrast, CDK12 inhibition triggers IPA. Finally, we show that dynamic O-GlcNAcylation predominantly interplays with CDK9: OGT inhibition augments CDK9 inhibitor effects on mRNA maturation due to defects in transcription elongation, while OGA inhibition rescues mRNA maturation failure caused by targeting CDK9, but induces IPA. Conclusion We show that dynamic O-GlcNAcylation is a negative regulator of mRNA biosynthesis and propose that the addition and removal of the modification serve as quality control-steps to ascertain successful generation of mature mRNAs. Our work identifies unprecedented redundancy in the regulation of RNA Pol II, which increases resilience towards transcriptional stress, and also underscores the difficulty of targeting transcription to control cancer. Graphical Abstract