Search

Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two commonly mutated splicing factors in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired association of HTATSF1 with mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.GRAPHICAL ABSTRACTHIGHLIGHTS-Oncogenic mutations of SF3B1 and U2AF1 cause a gene-body RNAPII elongation defect-RNAPII transcription elongation defect leads to transcription replication conflicts, DNA damage response, and changes to chromatin organization and H3K4me3 marks-The transcription elongation defect is linked to disruption of the early spliceosome formation through impaired interaction of HTATSF1 with mutant SF3B1.-Changes to chromatin organization reveal potential therapeutic strategies by targeting the Sin3/HDAC pathway

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga

Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage., Boddu et al combined AAV-intron trap, CRISPR/Cas9 and inducible Cre-recombinase systems to introduce the oncogenic K700E mutation in SF3B1, which is a splicing factor commonly mutated in multiple cancers, with more than 90% efficiency. Their approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes are detrimental to cell culture growth.

Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that promote efficient biogenesis of RNA-protein complexes. Depletion of the CB scaffolding protein coilin is lethal for vertebrate embryogenesis, making CBs a strong model for understanding the structure and function of MLOs. Although it is assumed that CBs form through biomolecular condensation, the biochemical and biophysical principles that govern CB dynamics have eluded study. Here, we identify features of the coilin protein that drive CB assembly and shape. Focusing on coilin’s N-terminal domain (NTD), we discovered its unexpected capacity for oligomerization in vivo. Single amino acid mutational analysis of coilin revealed distinct molecular interactions required for oligomerization and binding to the Nopp140 ligand, which facilitates CB assembly. We demonstrate that the intrinsically disordered regions of Nopp140 have substantial condensation properties and suggest that Nopp140 binding thereby remodels stable coilin oligomers to form a particle that recruits other functional components.

Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

Living cells, tissues and organisms are open, metabolically active systems that constantly exchange matter and energy with their environment in the form of heat. The heat exchanged is equal to the net enthalpy of all chemical reactions taking place within the system. Thus, heat dissipation can inform on the energetic costs of the constellation of cellular processes that contribute to physiology and address unanswered questions about development, responses to the environment, signaling and metabolic pathways, and the roles of morphological substructures. Here, we describe the methods we established to measure the heat dissipated by early zebrafish embryos undergoing synchronous cell cycles of cleavage stage embryogenesis, using isothermal calorimetry. The non-invasive nature of calorimetry and the versatility of these methods enables the investigation of the energetic costs of embryonic development and of the cellular processes associated with the early embryonic cell cycles.

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3’ untranslated regions (3’UTRs) of mRNA, but the identity of functional targets remains unknown. Here we used iCLIP to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of target mRNAs largely in 3’UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more “U/TAG” motifs compared to targets bound non-productively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute MSI2 loss. Eukaryotic elongation factor 3A (EIF3A) was determined to be an immediate, direct target. We propose that MSI2 down-regulation of EIF3A amplifies these effects on the proteome. Our results also underscore the challenges in defining functional targets of RBP since mere binding does not imply a discernible functional interaction.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2in vitro. Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (∼13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.

Living cells, tissues and organisms are open, metabolically active systems that constantly exchange matter and energy with their environment in the form of heat. The heat exchanged is equal to the net enthalpy of all chemical reactions taking place within the system. Thus, heat dissipation can inform on the energetic costs of the constellation of cellular processes that contribute to physiology and address unanswered questions about development, responses to the environment, signaling and metabolic pathways, and the roles of morphological substructures. Here, we describe the methods we established to measure the heat dissipated by early zebrafish embryos undergoing synchronous cell cycles of cleavage stage embryogenesis, using isothermal calorimetry. The non-invasive nature of calorimetry and the versatility of these methods enables the investigation of the energetic costs of embryonic development and of the cellular processes associated with the early embryonic cell cycles.

Long read sequencing technologies now allow high-quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide-resolution information about co-transcriptional RNA processing events-e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete poly(A)+ RNA and rRNA, and, finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Subcellular fractionation Basic Protocol 2: Nascent RNA isolation and adapter ligation Basic Protocol 3: cDNA amplicon preparation.

SummaryBiomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the ‘survival of motor neuron protein’ (SMN) is required for the formation of three different membraneless organelles (MLOs), we hypothesized that at least one region of SMN employs a unifying mechanism of condensation. Unexpectedly, we show here that SMN’s globular tudor domain was sufficient for dimerization-induced condensationin vivo, while its two intrinsically disordered regions (IDRs) were not. The condensate-forming property of the SMN tudor domain required binding to its ligand, dimethylarginine (DMA), and was shared by at least seven additional tudor domains in six different proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs – gems and Cajal bodies – were separate or overlapping. These findings show that the combination of a tudor domain bound to its DMA ligand – DMA-tudor – represents a versatile yet specific interaction module that regulates MLO assembly and defines their composition.

Thousands of genes produce polyadenylated mRNAs that still contain one or more introns. These transcripts are known as retained intron RNAs (RI-RNAs). In the past 10 years, RI-RNAs have been linked to post-transcriptional alternative splicing in a variety of developmental contexts, but they can also be dead-end products fated for RNA decay. Here we discuss the role of intron retention in shaping gene expression programs, as well as recent evidence suggesting that the biogenesis and fate of RI-RNAs is regulated by nuclear organization. We discuss the possibility that proximity of RNA to nuclear speckles - biomolecular condensates that are highly enriched in splicing factors and other RNA binding proteins - is associated with choices ranging from efficient co-transcriptional splicing, export and stability to regulated post-transcriptional splicing and possible vulnerability to decay.

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3’ end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and PRO-seq during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75 – 300 nucleotides of 3’ splice sites (3’SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3’ end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3’SS improves both splicing and 3’ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

Eukaryotic gene expression requires the cumulative activity of multiple molecular machines to synthesize and process newly transcribed pre-messenger RNA. Introns, the noncoding regions in pre-mRNA, must be removed by the spliceosome, which assembles on the pre-mRNA as it is transcribed by RNA polymerase II (Pol II). The assembly and activity of the spliceosome can be modulated by features including the speed of transcription elongation, chromatin, post-translational modifications of Pol II and histone tails, and other RNA processing events like 5'-end capping. Here, we review recent work that has revealed cooperation and coordination among co-transcriptional processing events and speculate on new avenues of research. We anticipate new mechanistic insights capable of unraveling the relative contribution of coupled processing to gene expression.

Summary The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance., Graphical Abstract, Highlights • Connecticut’s COVID-19 outbreak resulted from multiple domestic virus introductions • SARS-CoV-2 genomic data indicate that coast-to-coast spread occurred in the United States • Risk of introduction by domestic air travel exceeded international travel in March • Restrictions on international travel did not significantly alter risk estimates, Using genomics and air travel information, the spread of SARS-CoV-2 in the United States from coast to coast is shown to be more a consequence of domestic introductions than of international travel.

Nascent RNA sequencing has revealed that pre-mRNA splicing can occur shortly after introns emerge from RNA polymerase II (RNA Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here, we use single-molecule intron tracking (SMIT) to identify a cohort of regulators by machine learning in budding yeast. Of these, Nab2 displays reduced co-transcriptional splicing when depleted. Unexpectedly, these splicing defects are attributable to aberrant "intrusive" transcriptional readthrough from upstream genes, as revealed by long-read sequencing. Transcripts that originate from the intron-containing gene's own transcription start site (TSS) are efficiently spliced, indicating no direct role of Nab2 in splicing per se. This work highlights the coupling between transcription, splicing, and 3' end formation in the context of gene organization along chromosomes. We conclude that Nab2 is required for proper 3' end processing, which ensures gene-specific control of co-transcriptional RNA processing.

How do early embryos allocate the resources stored in the sperm and egg? Recently, we established isothermal calorimetry to measure heat dissipation by living zebra-fish embryos and to estimate the energetics of specific developmental events. During the reductive cleavage divisions, the rate of heat dissipation increases from ∼60 nJ · s-1 at the two-cell stage to ∼90 nJ · s-1 at the 1024-cell stage. Here we ask which cellular process(es) drive this increasing energetic cost. We present evidence that the cost is due to the increase in the total surface area of all the cells of the embryo. First, embryo volume stays constant during the cleavage stage, indicating that the increase is not due to growth. Second, the heat increase is blocked by nocodazole, which inhibits DNA replication, mitosis, and cell division; this suggests some aspect of cell proliferation contributes to these costs. Third, the heat increases in proportion to the total cell surface area rather than total cell number. Fourth, the heat increase falls within the range of the estimated costs of maintaining and assembling plasma membranes and associated proteins. Thus, the increase in total plasma membrane associated with cell proliferation is likely to contribute appreciably to the total energy budget of the embryo.

Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.

Several macromolecular machines collaborate to produce eukaryotic messenger RNA. RNA polymerase II (Pol II) translocates along genes that are up to millions of base pairs in length and generates a flexible RNA copy of the DNA template. This nascent RNA harbours introns that are removed by the spliceosome, which is a megadalton ribonucleoprotein complex that positions the distant ends of the intron into its catalytic centre. Emerging evidence that the catalytic spliceosome is physically close to Pol II in vivo implies that transcription and splicing occur on similar timescales and that the transcription and splicing machineries may be spatially constrained. In this Review, we discuss aspects of spliceosome assembly, transcription elongation and other co-transcriptional events that allow the temporal coordination of co-transcriptional splicing.

SR proteins connect nuclear pre-mRNA processing to mRNA export and translation. Botti et al. develop a quantitative nucleocytoplasmic shuttling assay and show that SR proteins are differentially modified and active in differentiated and pluripotent cells., SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein–protein and protein–RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.

Spliceosomal gene mutations function as drivers of hematologic malignancies and other cancers with an occurrence of more than 50% in myelodysplastic syndromes and secondary acute myeloid leukemia. Hotspot mutations S34F and Q157R in the two zinc finger domains of the splicing factor U2AF1, forming with U2AF2 the U2AF complex that recognizes 3' splice site (3'SS) of U2 introns, alter exon usage in a sequence-specific manner. However, how pathological U2AF1 mutations disrupt ordered splicing, from binding to recruitment of cooperating RNA binding proteins and ultimately splicing kinetics, is still not known at the molecular level. To obtain unique insights into in vivo RNA binding mechanisms, we performed fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA sequencing (freCLIP-seq) on human erythroleukemia (HEL) cells expressing wild-type (WT) or mutant (S34F, Q157R) U2AF1. Transcriptome-wide analysis of binding at single nucleotide resolution in light and heavy fractions, corresponding respectively to U2AF1 only and U2AF complex, allowed to: i) deconvolute U2AF1 signal peaking over the AG dinucleotide at the intronic end of the 3'SS region, and U2AF2 signal sitting on the adjacent polypyrimidine tract (PPT); ii) identify conformational changes in mutant U2AF1 binding with a novel peak in position -3 of the 3'SS region for S34F and in position +1 for Q157R. Alternative splicing analysis on newly collected RNA-seq data showed that less included exons present higher probability of U in position -3 for S34F and A in position +1 for Q157R, pinpointing a match with nucleotide positions affected by aberrant binding in freCLIP-seq. In both U2AF1 mutants, aberrant binding and splicing mechanisms affected genes involved in mRNA processing and transport (P-value We then performed a combined analysis of differential binding and aberrant splicing in U2AF1 mutants vs WT considering 4 categories: ">inclusion/>binding", "binding", ">inclusion/binding" with 123 events out of 309 (Figure 1A). Moreover, differential binding was not dependent on specific nucleotides in position -3: events characterized by increased S34F binding (Figure 1B, top), as well as events characterized by decreased S34F binding (Figure 1B, bottom), showed -3U in less included exons or -3C in more included exons. The binding analysis across the 4 categories showed that increased S34F binding was associated with reduced U2AF2 binding (Figure 1C, top) particularly in less included exons, while decreased S34F binding was associated with increased U2AF2 binding (Figure 1C, bottom) especially in more included exons. Finally, analysis of branch point and splice junction features revealed that PPT strength influences the splicing outcome with "binding" category characterized by a weak PPT that impairs U2AF2 binding in the presence of skewed U2AF1 S34F binding (Figure 1D). Additionally, transcriptome-wide RNA kinetics analysis by TimeLapse-seq demonstrated that U2AF1 S34F and Q157R, compared to WT, globally decrease synthesis of aberrantly spliced and bound 3'SS regions. Of note, this shutdown effect was particularly evident in the downstream exons pointing towards a role of U2AF1 mutations in a widespread alteration of RNA synthesis and splicing dynamics. Collectively, these results disclose novel molecular mechanisms of pathogenic U2AF1 mutations in the context of myeloid malignancies and provide the basis for the development of effective U2AF1 directed therapeutic strategies. Disclosures Hunck: Boehringer Ingelheim Fonds: Other: MD Fellowship.

How do early embryos apportion the resources stored in the sperm and egg? Recently, we established isothermal calorimetry (ITC) to measure heat dissipation by living zebrafish embryos and to estimate the energetics of specific developmental events. During the reductive cleavage divisions, the rate of heat dissipation increases from ∼60 nJ·s−1at the 2-cell stage to ∼90 nJ·s−1at the 1024-cell stage. Here we ask, which cellular process(es) drive these increasing energetic costs? We present evidence that the cost is due to the increase in the total surface area of all of the cells of the embryo. First, embryo volume stays constant during the cleavage stage, indicating that the increase is not due to growth. Second, the heat increase is blocked by nocodazole, which inhibits DNA replication, mitosis and cell division; this implicates some aspect of cell proliferation contributing to these costs. Third, the heat increase scales with total cell surface area rather than total cell number. Finally, the calculated costs of maintaining and assembling plasma membranes and associated proteins probably accounts for a significant proportion of the heat increase. Thus, the cell’s membrane is likely to contribute significantly to the total energy budget of the embryo.Highlight summary for TOCRodenfels et al. measure the energetic costs of early zebrafish development, using calorimetry. Embryonic heat dissipation increases, but, more slowly than the number of cells during early cleavage stage development. Instead, the heat dissipation scales with the energetic cost associated with maintaining and producing new plasma membrane.

At each active protein-encoding gene, nascent RNA is tethered to the DNA axis by elongating RNA polymerase II (Pol II) and is continuously altered by splicing and other processing events during its synthesis. This review discusses the development of three major methods that enable us to track the conversion of precursor messenger RNA (pre-mRNA) to messenger RNA (mRNA) products in vivo: live-cell imaging, metabolic labeling of RNA, and RNA-seq of purified nascent RNA. These approaches are complementary, addressing distinct issues of transcription rates and intron lifetimes alongside spatial information regarding the gene position of Pol II at which spliceosomes act. The findings will be placed in the context of active transcription units, each of which-because of the presence of nascent RNA, Pol II, and features of the chromatin environment-will recruit a potentially gene-specific constellation of RNA binding proteins and processing machineries.

Summary Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs—gems and Cajal bodies—were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules—combinations of tudor domains bound to their DMA ligand(s)—represent versatile yet specific regulators of MLO assembly, composition, and morphology.

Folding of RNA into secondary structures through intramolecular base pairing determines an RNA’s three-dimensional architecture and associated function. Simple RNA structures like stem loops can provide specialized functions independent of coding capacity, such as protein binding, regulation of RNA processing and stability, stimulation or inhibition of translation. RNA catalysis is dependent on tertiary structures found in the ribosome, tRNAs and group I and II introns. While the extent to which non-coding RNAs contribute to cellular maintenance is generally appreciated, the fact that both non-coding and coding RNA can assume relevant structural states has only recently gained attention. In particular, the co-transcriptional folding of nascent RNA of all classes has the potential to regulate co-transcriptional processing, RNP (ribonucleoprotein particle) formation, and transcription itself. Riboswitches are established examples of co-transcriptionally folded coding RNAs that directly regulate transcription, mainly in prokaryotes. Here we discuss recent studies in both prokaryotes and eukaryotes showing that structure formation may carry a more widespread regulatory logic during RNA synthesis. Local structures forming close to the catalytic center of RNA polymerases have the potential to regulate transcription by reducing backtracking. In addition, stem loops or more complex structures may alter co-transcriptional RNA processing or its efficiency. Several examples of functional structures have been identified to date, and this review provides an overview of physiologically distinct processes where co-transcriptionally folded RNA plays a role. Experimental approaches such as single-molecule FRET and in vivo structural probing to further advance our insight into the significance of co-transcriptional structure formation are discussed.

Background Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood. Methods We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Findings The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection in vitro. To better understand potential immune mechanisms shielding placental cells from infection in vivo, we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia. Conclusions SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. Funding NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center., Graphical abstract, Context and significance Pregnant women with COVID-19 are at increased risk for severe illness and pregnancy complications compared with non-pregnant women. Researchers at Yale School of Medicine analyzed placentas from SARS-CoV-2-infected women at the time of delivery and found that, although placental cells are susceptible to infection in vitro, viral RNA is rarely detected in clinical samples. The Yale team observed local immune responses at the maternal-fetal interface, including upregulation of interferon pathways and activation of T and NK cells. Although placental immune activation during maternal SARS-CoV-2 infection likely represents a host defense mechanism of shielding the maternal-fetal interface from infection, these inflammatory changes may contribute to the increased risk for complications seen in COVID-19-affected pregnancies., COVID-19 is more severe in pregnant women and can lead to adverse fetal outcomes. Through histological and gene expression studies of placentas from infected women, Lu-Culligan et al. find that maternal SARS-CoV-2 infection during term pregnancy and delivery is associated with immune activation at the maternal-fetal interface even in the absence of detectable virus in the placenta.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA—the most highly expressed transcript in erythroblasts—was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing.

During gene expression, protein-coding transcripts are shaped by multiple processing events: 5' end capping, pre-mRNA splicing, RNA editing, and 3' end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3' end cleavage or post-transcriptionally. Pre-mRNA splicing - the removal of introns and ligation of exons - can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.

Following fertilization, embryos develop for a substantial amount of time with a transcriptionally silent genome. Thus, early development is maternally programmed, as it solely relies on RNAs and proteins that are provided by the female gamete. However, these maternal instructions are not sufficient to support later steps of embryogenesis and are therefore gradually replaced by novel products synthesized from the zygotic genome. This switch in the origin of molecular players that drive early development is known as the maternal-to-zygotic transition (MZT). MZT is a universal phenomenon among all metazoans and comprises two interconnected processes: maternal mRNA degradation and the transcriptional awakening of the zygotic genome. The recent adaptation of high-throughput methods for use in embryos has deepened our knowledge of the molecular principles underlying MZT. These mechanisms comprise conserved strategies for RNA regulation that operate in many well-studied cellular contexts but that have adapted differently to early development. In this Review, we will discuss advances in our understanding of post-transcriptional regulatory pathways that drive maternal mRNA clearance during MZT, with an emphasis on recent data in zebrafish embryos on codon-mediated mRNA decay, the contributions of microRNAs (miRNAs) and RNA-binding proteins to this process, and the roles of RNA modifications in the stability control of maternal mRNAs.

The Cajal body (CB) is a nuclear compartment enriched in specific protein and RNA components without the barrier of a lipid bilayer. Like the nucleolus in the nucleus and stress granules in the cyt...

A decade ago, we believed that at least 300 RNA binding proteins (RBPs) were encoded in our genomes based on annotations of known or predicted RNA binding domains. Deciphering the roles of those RBPs in regulated gene expression was a vast frontier awaiting exploration. Since then, the field has developed a number of key tools that navigate the landscape of cellular RNA. These rely principally on UV crosslinking to create covalent bonds between RBPs and target RNAs in vivo , revealing not only target identities but also local binding sites upon RNA-Seq. More recently, a reverse protocol – mRNA interactome capture – has enabled the identification of the proteins that interact with mRNA. Astonishingly, the number of RBPs has grown to more than 1000, and we must now understand what they do. Here, we discuss the application of these methods to model organisms, focusing on the zebrafish Danio rerio , which provide unique biological contexts for the analysis of RBPs and their functions.

A gene's “expression profile” denotes the number of transcripts present relative to all other transcripts. The overall rate of transcript production is determined by transcription and RNA processing rates. While the speed of elongating RNA polymerase II has been characterized for many different genes and organisms, gene-architectural features – primarily the number and length of exons and introns – have recently emerged as important regulatory players. Several new studies indicate that rapidly cycling cells constrain gene-architecture toward short genes with a few introns, allowing efficient expression during short cell cycles. In contrast, longer genes with long introns exhibit delayed expression, which can serve as timing mechanisms for patterning processes. These findings indicate that cell cycle constraints drive the evolution of gene-architecture and shape the transcriptome of a given cell type. Furthermore, a tendency for short genes to be evolutionarily young hints at links between cellular constraints and the evolution of animal ontogeny.

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

All living systems function out of equilibrium and exchange energy in the form of heat with their environment. Thus, heat flow can inform on the energetic costs of cellular processes, which are largely unknown. Here, we have repurposed an isothermal calorimeter to measure heat flow between developing zebrafish embryos and the surrounding medium. Heat flow increased over time with cell number. Unexpectedly, a prominent oscillatory component of the heat flow, with periods matching the synchronous early reductive cleavage divisions, persisted even when DNA synthesis and mitosis were blocked by inhibitors. Instead, the heat flow oscillations were driven by the phosphorylation and dephosphorylation reactions catalyzed by the cell-cycle oscillator, the biochemical network controlling mitotic entry and exit. We propose that the high energetic cost of cell-cycle signaling reflects the significant thermodynamic burden of imposing accurate and robust timing on cell proliferation during development.