RESUMEN
Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. Here, we use transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, we characterize the in vivo function of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. Our study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo.
Asunto(s)
Drosophila/fisiología , Insulina/fisiología , Longevidad/genética , ARN Circular/fisiología , Envejecimiento , Animales , Animales Modificados Genéticamente , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Masculino , Mutación , Neuronas/fisiología , Sulfotransferasas/genética , TranscriptomaRESUMEN
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.
Asunto(s)
Cromatina , Proteínas Musculares , Desaminasas APOBEC/genética , Desaminasas APOBEC-1/genética , Diferenciación Celular/genética , Cromatina/genética , Citidina Desaminasa/metabolismo , ADN , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/metabolismo , ARN Mensajero/genética , Animales , RatonesRESUMEN
The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding-deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault-tolerant, functionally redundant NMD activators in human cells.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Unión al ARN , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
Ageing is a complex process with common and distinct features across tissues. Unveiling the underlying processes driving ageing in individual tissues is indispensable to decipher the mechanisms of organismal longevity. Caenorhabditis elegans is a well-established model organism that has spearheaded ageing research with the discovery of numerous genetic pathways controlling its lifespan. However, it remains challenging to dissect the ageing of worm tissues due to the limited description of tissue pathology and access to tissue-specific molecular changes during ageing. In this study, we isolated cells from five major tissues in young and old worms and profiled the age-induced transcriptomic changes within these tissues. We observed a striking diversity of ageing across tissues and identified different sets of longevity regulators therein. In addition, we found novel tissue-specific factors, including irx-1 and myrf-2, which control the integrity of the intestinal barrier and sarcomere structure during ageing respectively. This study demonstrates the complexity of ageing across worm tissues and highlights the power of tissue-specific transcriptomic profiling during ageing, which can serve as a resource to the field.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Longevidad/genética , TranscriptomaRESUMEN
The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation.
Asunto(s)
Benchmarking , ARN Circular , Humanos , ARN Circular/genética , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use general established rules. We present a dataset with 4 cell lines and combinations for SMG5, SMG6 and SMG7 knockdowns or SMG7 knockout. Based on this dataset, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information from Ensembl, Gencode consensus RiboSeq ORFs and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which, 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon and 6,433 are significantly upregulated in three or more comparisons of NMD active vs deficient cell lines. We present an in-depth view on these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.
RESUMEN
Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.
Asunto(s)
Empalme Alternativo/fisiología , Exones/fisiología , Sitios de Empalme de ARN/fisiología , Secuencia de Consenso/genética , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Intrones , Precursores del ARN/fisiología , Empalme del ARN/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Transcriptoma/genéticaRESUMEN
Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.
Asunto(s)
Enfermedad de Huntington , Ratones , Animales , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , ARN Circular/genética , Empalme del ARN , Empalme Alternativo/genética , Perfilación de la Expresión Génica , Expansión de Repetición de Trinucleótido/genética , Proteína Huntingtina/genéticaRESUMEN
N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
Asunto(s)
Adenosina/análogos & derivados , Axones/fisiología , Proteínas de Drosophila/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuronas/citología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Neuronas/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Nanopore long-read sequencing enables real-time monitoring and controlling of individual nanopores. This allows us to enrich or deplete specific sequences in DNA sequencing in a process called "adaptive sampling." So far, adaptive sampling (AS) was not applicable to the direct sequencing of RNA. Here, we show that AS is feasible and useful for direct RNA sequencing (DRS), which has its specific technical and biological challenges. Using a well-controlled in vitro transcript-based model system, we identify essential characteristics and parameter settings for AS in DRS, as the superior performance of depletion over enrichment. Here, the efficiency of depletion is close to the theoretical maximum. Additionally, we demonstrate that AS efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our AS approach to poly(A)-enriched RNA samples from human-induced pluripotent stem cell-derived cardiomyocytes and mouse whole heart tissue and show efficient 2.5- to 2.8-fold depletion of highly abundant mitochondrial-encoded transcripts. Finally, we characterize depletion and enrichment performance for complex transcriptome subsets, that is, at the level of the entire Chromosome 11, proving the general applicability of direct RNA AS. Our analyses provide evidence that AS is especially useful to enable the detection of lowly expressed transcripts and reduce the sequencing of highly abundant disturbing transcripts.
Asunto(s)
Nanoporos , ARN , Humanos , Animales , Ratones , ARN/genética , Análisis de Secuencia de ARN , ARN Mensajero/genética , Transcriptoma/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.
Asunto(s)
Nucleósido Q , ARN de Transferencia , Anticodón/genética , Escherichia coli/genética , Eucariontes/genética , Nucleósido Q/análisis , ARN , ARN de Transferencia/química , Schizosaccharomyces/química , Schizosaccharomyces/genética , Análisis de Secuencia de ARNRESUMEN
Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.
Asunto(s)
Biosíntesis de Proteínas , Transcriptoma , Humanos , Arsenitos/toxicidad , Proteómica , ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Circular RNAs are an endogenous long-lived and abundant noncoding species. Despite their prevalence, only a few circRNAs have been dissected mechanistically to date. Here, we cataloged nascent RNA-enriched circRNAs from primary human cells and functionally assigned a role to circRAB3IP in sustaining cellular homeostasis. We combined "omics" and functional experiments to show how circRAB3IP depletion deregulates hundreds of genes, suppresses cell cycle progression, and induces senescence-associated gene expression changes. Conversely, excess circRAB3IP delivered to endothelial cells via extracellular vesicles suffices for accelerating their division. We attribute these effects to an interplay between circRAB3IP and the general splicing factor SF3B1, which can affect transcript variant expression levels of cell cycle-related genes. Together, our findings link the maintenance of cell homeostasis to the presence of a single circRNA.
Asunto(s)
MicroARNs , ARN Circular , Humanos , ARN Circular/genética , Células Endoteliales/metabolismo , Proliferación Celular/genética , ARN Mensajero/genética , Expresión Génica , MicroARNs/genéticaRESUMEN
MOTIVATION: Alternative RNA splicing plays a crucial role in defining protein function. However, despite its relevance, there is a lack of tools that characterize effects of splicing on protein interaction networks in a mechanistic manner (i.e. presence or absence of protein-protein interactions due to RNA splicing). To fill this gap, we present Linear Integer programming for Network reconstruction using transcriptomics and Differential splicing data Analysis (LINDA) as a method that integrates resources of protein-protein and domain-domain interactions, transcription factor targets, and differential splicing/transcript analysis to infer splicing-dependent effects on cellular pathways and regulatory networks. RESULTS: We have applied LINDA to a panel of 54 shRNA depletion experiments in HepG2 and K562 cells from the ENCORE initiative. Through computational benchmarking, we could show that the integration of splicing effects with LINDA can identify pathway mechanisms contributing to known bioprocesses better than other state of the art methods, which do not account for splicing. Additionally, we have experimentally validated some of the predicted splicing effects that the depletion of HNRNPK in K562 cells has on signalling.
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Empalme Alternativo , Mapas de Interacción de Proteínas , Empalme del ARN , Benchmarking , Análisis de DatosRESUMEN
BACKGROUND: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1. METHODS: Different Adar1-mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis. RESULTS: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7. Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1-mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1-deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes. CONCLUSIONS: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.
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Adenosina Desaminasa , Insuficiencia Cardíaca , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Inosina/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/metabolismo , Ratones , Ratones Mutantes , FN-kappa B/metabolismo , ARNRESUMEN
BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.
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Proteínas de Unión al ADN , Células Endoteliales , Endotelio Vascular , ARN Circular , Factores de Transcripción , Animales , Humanos , Ratones , Circulación Sanguínea , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Unión Proteica , ARN Circular/genética , ARN Circular/metabolismo , Sindecano-4/metabolismo , Factores de Transcripción/genéticaRESUMEN
The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing and nonsense-mediated mRNA decay (NMD) via various interacting proteins. The peripheral EJC-binding protein RNPS1 was reported to serve two insufficiently characterized functions: suppressing mis-splicing of cryptic splice sites and activating NMD in the cytoplasm. The analysis of transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines supports the conclusion that RNPS1 can moderately influence NMD activity, but is not a globally essential NMD factor. However, numerous aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that the RRM and C-terminal domain of RNPS1 both contribute partially to regulate RNPS1-dependent splicing events. We defined the RNPS1 core interactome using complementary immunoprecipitations and proximity labeling, which identified interactions with splicing-regulatory factors that are dependent on the C-terminus or the RRM domain of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.
Asunto(s)
Ribonucleoproteínas , Transcriptoma , Exones , Humanos , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
BACKGROUND: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. METHODS: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. RESULTS: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes. No mononucleated/diploid 15N-thymidine-labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. CONCLUSIONS: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.
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Miocitos Cardíacos , Condicionamiento Físico Animal , Animales , Calcineurina/metabolismo , Humanos , Lactante , Ratones , Miocitos Cardíacos/citología , Timidina/metabolismoRESUMEN
Adenosine-to-inosine RNA editing and pre-mRNA splicing largely occur cotranscriptionally and influence each other. Here, we use mice deficient in either one of the two editing enzymes ADAR (ADAR1) or ADARB1 (ADAR2) to determine the transcriptome-wide impact of RNA editing on splicing across different tissues. We find that ADAR has a 100× higher impact on splicing than ADARB1, although both enzymes target a similar number of substrates with a large common overlap. Consistently, differentially spliced regions frequently harbor ADAR editing sites. Moreover, catalytically dead ADAR also impacts splicing, demonstrating that RNA binding of ADAR affects splicing. In contrast, ADARB1 editing sites are found enriched 5' of differentially spliced regions. Several of these ADARB1-mediated editing events change splice consensus sequences, therefore strongly influencing splicing of some mRNAs. A significant overlap between differentially edited and differentially spliced sites suggests evolutionary selection toward splicing being regulated by editing in a tissue-specific manner.
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Adenosina Desaminasa/genética , Edición de ARN/genética , Procesamiento Postranscripcional del ARN/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Adenosina/química , Animales , Inosina/química , Ratones , Ratones Noqueados , ARN Circular/genética , ARN Mensajero/genética , Análisis de Secuencia de ARNRESUMEN
The current ecosystem of single cell RNA-seq platforms is rapidly expanding, but robust solutions for single cell and single molecule full- length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single cell sequencing method on the 10x Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g. for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing satura- tion is low. ScNapBar uses unique molecular identifier (UMI) or Naive Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore datasets. We further applied ScNapBar to pools of cells with an active or a silenced non-sense mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.