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Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.
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Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.
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Resistencia a Antineoplásicos , Neoplasias Hematológicas , Proteínas de Unión al ARN , Humanos , Resistencia a Antineoplásicos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/genética , Elementos Ricos en Adenilato y Uridilato , Regulación Neoplásica de la Expresión Génica , Animales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Estabilidad del ARN , Unión ProteicaRESUMEN
Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.
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Células Procariotas , Transcripción Genética , Transcripción Genética/genética , Células Procariotas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Células Eucariotas/metabolismo , Humanos , Regulación de la Expresión Génica/genética , Animales , Eucariontes/genética , Empalme del ARN/genética , Procesamiento Postranscripcional del ARN/genética , ARN/metabolismo , ARN/genéticaRESUMEN
Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.
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Cromatina , Neoplasias , Animales , Humanos , Ratones , Cromatina/genética , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Empalme del ARN/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
Global warming is caused by human activity, such as the burning of fossil fuels, which produces high levels of greenhouse gasses. As a consequence, climate change impacts all organisms and the greater ecosystem through changing conditions from weather patterns to the temperature, pH and salt concentrations found in waterways and soil. These environmental changes fundamentally alter many parameters of the living world, from the kinetics of chemical reactions and cellular signaling pathways to the accumulation of unforeseen chemicals in the environment, the appearance and dispersal of new diseases, and the availability of traditional foods. Some organisms adapt to extremes, while others cannot. This article asks five questions that prompt us to consider the foundational knowledge that biochemistry can bring to the table as we meet the challenge of climate change. We approach climate change from the molecular point of view, identifying how cells and organisms - from microbes to plants and animals - respond to changing environmental conditions. To embrace the concept of "one health" for all life on the planet, we argue that we must leverage biochemistry, cell biology, molecular biophysics and genetics to fully understand the impact of climate change on the living world and to bring positive change.
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Excision of introns during splicing regulates gene expression. In this issue, work by Sung et al. (https://doi.org/10.1083/jcb.202111151) demonstrates that the timing of intron removal in response to stress is coordinated in nuclear speckles, adding a component of spatial regulation to co-/post-transcriptional splicing.
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Empalme Alternativo , Empalme del ARN , Intrones/genética , Empalme del ARN/genética , Motas NuclearesRESUMEN
Alfonso-Gonzalez et al.1 present an innovative combination of long-read-sequencing approaches that reveal coupling of alternative transcription start sites and alternative polyadenylation site usage on a global level.
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Poliadenilación , Regiones Promotoras GenéticasRESUMEN
Biomolecular condensates (BMCs) can facilitate or inhibit diverse cellular functions. BMC formation is driven by noncovalent protein-protein, protein-RNA, and RNA-RNA interactions. Here, we focus on Tudor domain-containing proteins - such as survival motor neuron protein (SMN) - that contribute to BMC formation by binding to dimethylarginine (DMA) modifications on protein ligands. SMN is present in RNA-rich BMCs, and its absence causes spinal muscular atrophy (SMA). SMN's Tudor domain forms cytoplasmic and nuclear BMCs, but its DMA ligands are largely unknown, highlighting open questions about the function of SMN. Moreover, DMA modification can alter intramolecular interactions and affect protein localization. Despite these emerging functions, the lack of direct methods of DMA detection remains an obstacle to understanding Tudor-DMA interactions in cells.
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Proteínas de Unión al ARN , ARN , Ligandos , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN/metabolismoRESUMEN
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. HIGHLIGHTS: Oncogenic mutations of SF3B1 and U2AF1 cause a gene-body RNAPII elongation defectRNAPII transcription elongation defect leads to transcription replication conflicts, DNA damage response, and changes to chromatin organization and H3K4me3 marksThe 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.
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Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back 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, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions.
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Imagenología Tridimensional , Programas Informáticos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodosRESUMEN
Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that concentrate and promote efficient biogenesis of snRNA-protein complexes involved in splicing (snRNPs). Depletion of the CB scaffolding protein coilin disperses snRNPs, making CBs a model system for studying the structure and function of MLOs. Although it is assumed that CBs form through condensation, the biomolecular interactions responsible remain elusive. Here, we discover the unexpected capacity of coilin's N-terminal domain (NTD) to form extensive fibrils in the cytoplasm and discrete nuclear puncta in vivo. Single amino acid mutational analysis reveals distinct molecular interactions between coilin NTD proteins to form fibrils and additional NTD interactions with the nuclear Nopp140 protein to form puncta. We provide evidence that Nopp140 has condensation capacity and is required for CB assembly. From these observations, we propose a model in which coilin NTD-NTD mediated assemblies make multivalent contacts with Nopp140 to achieve biomolecular condensation in the nucleus.
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Cuerpos Enrollados , Ribonucleoproteínas Nucleares Pequeñas , Aminoácidos/metabolismo , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Células HeLa , Humanos , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
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.
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ARN , Transcriptoma , Humanos , Nucleótidos , ARN/química , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Transcriptoma/genéticaRESUMEN
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.
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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.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Factor de Empalme U2AF , Gránulos de Estrés , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Síndromes Mielodisplásicos/genética , Sitios de Empalme de ARN , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismo , Gránulos de Estrés/metabolismoRESUMEN
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.
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Sistemas CRISPR-Cas/fisiología , Integrasas/fisiología , Intrones/fisiología , Neoplasias/fisiopatología , Roturas del ADN de Doble Cadena , Dependovirus , Recombinación Homóloga , Humanos , Neoplasias/enzimología , Neoplasias/genéticaRESUMEN
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.
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Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Animales , Arginina/metabolismo , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligandos , Metilación , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
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.
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Calorimetría/métodos , Embrión no Mamífero/fisiología , Pez Cebra/embriología , Animales , Ciclo Celular , Desarrollo Embrionario , Programas InformáticosRESUMEN
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.
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COVID-19 , Complicaciones Infecciosas del Embarazo , Enzima Convertidora de Angiotensina 2/genética , Femenino , Humanos , Placenta/metabolismo , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , SARS-CoV-2RESUMEN
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.