RESUMO
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared with nuclear mRNAs, mt-mRNAs were produced 1,100-fold more, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors LRPPRC and FASTKD5 identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
Assuntos
Mitocôndrias , Ribossomos Mitocondriais , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Fosforilação Oxidativa , Proteínas Mitocondriais/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismoRESUMO
Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
Assuntos
Núcleo Celular , Cromatina , RNA Helicases DEAD-box , RNA Mensageiro , Animais , Humanos , Camundongos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidade de RNA , Transporte Ativo do Núcleo Celular , Polirribossomos/metabolismo , Polirribossomos/genética , Aprendizado de Máquina , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Exossomos/metabolismo , Exossomos/genéticaRESUMO
Understanding how splicing events are coordinated across numerous introns in metazoan RNA transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of co-transcriptional processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. Long nano-COP reads reveal that, in human and Drosophila cells, splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B rapidly diminished global co-transcriptional splicing. We found that splicing order does not strictly follow the order of transcription and is associated with cis-acting elements, alternative splicing, and RNA-binding factors. Further, neighboring introns in human cells tend to be spliced concurrently, implying that splicing of these introns occurs cooperatively. Thus, nano-COP unveils the organizational complexity of RNA processing.
Assuntos
Sequenciamento por Nanoporos , Nanoporos , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Íntrons , Células K562 , Cinética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).
Assuntos
Ataxia Cerebelar , Expansão das Repetições de DNA , Íntrons , Humanos , Austrália , Canadá , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Íntrons/genética , Expansão das Repetições de DNA/genéticaRESUMO
OBJECTIVE: We introduce a non-invasive MR-Acoustic Radiation Force Imaging (ARFI)-based elastography method that provides both the local shear modulus and temperature maps for the monitoring of High Intensity Focused Ultrasound (HIFU) therapy. MATERIALS AND METHODS: To take tissue anisotropy into account, the local shear modulus µ is determined in selected radial directions around the focal spot by fitting the phase profiles to a linear viscoelastic model, including tissue-specific mechanical relaxation time τ. MR-ARFI was evaluated on a calibrated phantom, then applied to the monitoring of HIFU in a gel phantom, ex vivo and in vivo porcine muscle tissue, in parallel with MR-thermometry. RESULTS: As expected, the shear modulus polar maps reflected the isotropy of phantoms and the anisotropy of muscle. In the HIFU monitoring experiments, both the shear modulus polar map and the thermometry map were updated with every pair of MR-ARFI phase images acquired with opposite MR-ARFI-encoding. The shear modulus was found to decrease (phantom and ex vivo) or increase (in vivo) during heating, before remaining steady during the cooling phase. The mechanical relaxation time, estimated pre- and post-HIFU, was found to vary in muscle tissue. DISCUSSION: MR-ARFI allowed for monitoring of viscoelasticity changes around the HIFU focal spot even in anisotropic muscle tissue.
Assuntos
Ablação por Ultrassom Focalizado de Alta Intensidade , Imageamento por Ressonância Magnética , Animais , Suínos , Anisotropia , Imageamento por Ressonância Magnética/métodos , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Espectroscopia de Ressonância Magnética , AcústicaRESUMO
RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554AâG (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.
Assuntos
Regulação para Baixo/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Mutação , RNA Polimerase III/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Genes Recessivos , Células HeLa , HumanosRESUMO
Pseudouridylation is a common post-transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity-biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post-transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine-Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.
Assuntos
Sobrevivência Celular , Transferases Intramoleculares/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas de Transporte , Linhagem Celular , Humanos , Ligação Proteica , Transporte Proteico , RNA/genética , RNA/metabolismo , Transporte de RNA , RNA Mitocondrial , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismoRESUMO
Congenital myopathies define a heterogeneous group of neuromuscular diseases with neonatal or childhood hypotonia and muscle weakness. The genetic cause is still unknown in many patients, precluding genetic counselling and better understanding of the physiopathology. To identify novel genetic causes of congenital myopathies, exome sequencing was performed in three consanguineous families. We identified two homozygous frameshift mutations and a homozygous nonsense mutation in the mitogen-activated protein triple kinase ZAK. In total, six affected patients carry these mutations. Reverse transcription polymerase chain reaction and transcriptome analyses suggested nonsense mRNA decay as a main impact of mutations. The patients demonstrated a generalized slowly progressive muscle weakness accompanied by decreased vital capacities. A combination of proximal contractures with distal joint hyperlaxity is a distinct feature in one family. The low endurance and compound muscle action potential amplitude were strongly ameliorated on treatment with anticholinesterase inhibitor in another patient. Common histopathological features encompassed fibre size variation, predominance of type 1 fibre and centralized nuclei. A peculiar subsarcolemmal accumulation of mitochondria pointing towards the centre of the fibre was a novel histological hallmark in one family. These findings will improve the molecular diagnosis of congenital myopathies and implicate the mitogen-activated protein kinase (MAPK) signalling as a novel pathway altered in these rare myopathies.
Assuntos
Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Miopatias Congênitas Estruturais , Proteínas Quinases/genética , Adulto , Consanguinidade , Exoma , Feminino , Humanos , MAP Quinase Quinase Quinases , Masculino , Mutação , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , LinhagemRESUMO
Mutations in GALC cause Krabbe disease. This autosomal recessive leukodystrophy generally presents in early infancy as a severe disorder, but sometimes manifests as a milder adult-onset disease with spastic paraplegia as the main symptom. We recruited a family with five affected individuals presenting with adult-onset predominant cerebellar ataxia with mild spasticity. Whole exome sequencing (WES) revealed one novel and one previously reported compound heterozygous variants in GALC. Magnetic resonance imaging (MRI) confirmed the presence of typical Krabbe features. Our findings expand the phenotypic spectrum of adult-onset Krabbe disease and demonstrate the usefulness of combining WES and pattern-specific MRI for the diagnosis of neurodegenerative diseases.
Assuntos
Ataxia Cerebelar/genética , Galactosilceramidase/genética , Leucodistrofia de Células Globoides/genética , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Adulto JovemRESUMO
Episodic ataxias (EAs) are a heterogeneous group of neurological disorders characterized by recurrent attacks of ataxia. Mutations in KCNA1 and CACNA1A account for the majority of EA cases worldwide. We recruited a two-generation family affected with EA of unknown subtype and performed whole-exome sequencing on two affected members. This revealed a novel heterozygous mutation c.211_212insA (p.I71NfsX27) leading to a premature stop codon in FGF14. Mutations in FGF14 are known to cause spinocerebellar ataxia type 27 (SCA27). Sanger sequencing confirmed segregation within the family. Our findings expand the phenotypic spectrum of SCA27 by underlining the possible episodic nature of this ataxia.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Adulto , Ataxia/genética , Códon sem Sentido , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , LinhagemRESUMO
Mutations in POLR3A encoding the largest subunit of RNA polymerase III (Pol III) were found to be responsible for the majority of cases presenting with three clinically overlapping hypomyelinating leukodystrophy phenotypes. We uncovered in three cases without POLR3A mutation recessive mutations in POLR3B, which codes for the second largest subunit of Pol III. Mutations in genes coding for Pol III subunits are a major cause of childhood-onset hypomyelinating leukodystrophies with prominent cerebellar dysfunction, oligodontia, and hypogonadotropic hypogonadism.
Assuntos
Códon sem Sentido/genética , Predisposição Genética para Doença/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Mutação de Sentido Incorreto/genética , RNA Polimerase III/genética , Sequência de Aminoácidos , Sequência de Bases , Cerebelo/patologia , Criança , Corpo Caloso/patologia , Genes Recessivos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Modelos Moleculares , Homologia de Sequência de AminoácidosRESUMO
Splicing is a highly regulated process critical for proper pre-mRNA maturation and the maintenance of a healthy cellular environment. Splicing events are impacted by ongoing transcription, neighboring splicing events, and cis and trans regulatory factors on the respective pre-mRNA transcript. Within this complex regulatory environment, splicing kinetics have the potential to influence splicing outcomes but have historically been challenging to study in vivo. In this review, we highlight recent technological advancements that have enabled measurements of global splicing kinetics and of the variability of splicing kinetics at single introns. We demonstrate how identifying features that are correlated with splicing kinetics has increased our ability to form potential models for how splicing kinetics may be regulated in vivo.
RESUMO
Objective.The aim of the paper is to propose an all-in-one method based on magnetic resonance-supersonic shear wave imaging (MR-SSI) and proton resonance frequency shift (PRFS) to monitor high intensity focused ultrasound (HIFU) thermal ablations.Approach.Mechanical properties have been shown to be related to tissue damage induced by thermal ablations. Monitoring elasticity in addition to temperature changes may help in ensuring the efficacy and the accuracy of HIFU therapies. For this purpose, an MR-SSI method has been developed where the ultrasonic transducer is used for both mechanical wave generation and thermal ablation. Transient quasi-planar shear waves are generated using the acoustic radiation force, and their propagation is monitored in motion-sensitized phase MR images. Using a single-shot gradient-echo echo-planar-imaging sequence, MR images can be acquired at a sufficiently high temporal resolution to provide an update of PRFS thermometry and MR-SSI elastography maps in real time.Main results.The proposed method was first validated on a calibrated elasticity phantom, in which both the possibility to detect inclusions with different stiffness and repeatability were demonstrated. The standard deviation between the 8 performed measurements was 2% on the background of the phantom and 11%, at most, on the inclusions. A second experiment consisted in performing a HIFU heating in a gelatin phantom. The temperature increase was estimated to be 9 °C and the shear modulus was found to decrease from 2.9 to 1.8 kPa, reflecting the gel softening around the HIFU focus, whereas it remained steady in non-heated areas.Significance.The proposed MR-SSI technique allows monitoring HIFU ablations using thermometry and elastography simultaneously, without the need for an additional external mechanical exciter such as those used in MR elastography.
Assuntos
Técnicas de Imagem por Elasticidade , Tratamento por Ondas de Choque Extracorpóreas , Ablação por Ultrassom Focalizado de Alta Intensidade , Termometria , Técnicas de Imagem por Elasticidade/métodos , Termometria/métodos , Elasticidade , Ultrassom , Imageamento por Ressonância Magnética/métodos , Ablação por Ultrassom Focalizado de Alta Intensidade/métodosRESUMO
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared to nuclear mRNAs, mt-mRNAs were produced 700-fold higher, degraded 5-fold faster, and accumulated to 170-fold higher levels. Quantitative modeling and depletion of mitochondrial factors, LRPPRC and FASTKD5, identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
RESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells, in which gene expression must be coordinated across organelles using distinct pools of ribosomes. How cells produce and maintain the accurate subunit stoichiometries for these OXPHOS complexes remains largely unknown. To identify genes involved in dual-origin protein complex synthesis, we performed FACS-based genome-wide screens analyzing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of cytochrome c oxidase (Complex IV). We identified novel genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6 . We found that PREPL specifically regulates Complex IV biogenesis by interacting with mitochondrial protein synthesis machinery, while NME6, an uncharacterized nucleoside diphosphate kinase (NDPK), controls OXPHOS complex biogenesis through multiple mechanisms reliant on its NDPK domain. First, NME6 maintains local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Second, through stabilizing interactions with RCC1L, NME6 modulates the activity of mitoribosome regulatory complexes, leading to disruptions in mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression. Finally, we present these screens as a resource, providing a catalog of genes involved in mitonuclear gene regulation and OXPHOS biogenesis.
RESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) complexes are assembled from proteins encoded by both nuclear and mitochondrial DNA. These dual-origin enzymes pose a complex gene regulatory challenge for cells requiring coordinated gene expression across organelles. To identify genes involved in dual-origin protein complex synthesis, we performed fluorescence-activated cell-sorting-based genome-wide screens analysing mutant cells with unbalanced levels of mitochondrial- and nuclear-encoded subunits of Complex IV. We identified genes involved in OXPHOS biogenesis, including two uncharacterized genes: PREPL and NME6. We found that PREPL specifically impacts Complex IV biogenesis by acting at the intersection of mitochondrial lipid metabolism and protein synthesis, whereas NME6, an uncharacterized nucleoside diphosphate kinase, controls OXPHOS biogenesis through multiple mechanisms reliant on its NDPK domain. Firstly, NME6 forms a complex with RCC1L, which together perform nucleoside diphosphate kinase activity to maintain local mitochondrial pyrimidine triphosphate levels essential for mitochondrial RNA abundance. Secondly, NME6 modulates the activity of mitoribosome regulatory complexes, altering mitoribosome assembly and mitochondrial RNA pseudouridylation. Taken together, we propose that NME6 acts as a link between compartmentalized mitochondrial metabolites and mitochondrial gene expression.
Assuntos
DNA Mitocondrial , Núcleosídeo-Difosfato Quinase , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , RNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Regulação da Expressão Gênica , Fosforilação Oxidativa , Núcleosídeo-Difosfato Quinase/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.
Assuntos
Precursores de RNA , Splicing de RNA , Humanos , Íntrons/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Processamento Alternativo/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
BACKGROUND: Small nucleolar RNAs (snoRNAs) are abundant noncoding RNAs best known for their involvement in ribosomal RNA maturation. In mammals, most expressed snoRNAs are embedded in introns of longer genes and produced through transcription and splicing of their host. Intronic snoRNAs were long viewed as inert passengers with little effect on host expression. However, a recent study reported a snoRNA influencing the splicing and ultimate output of its host gene. Overall, the general contribution of intronic snoRNAs to host expression remains unclear. RESULTS: Computational analysis of large-scale human RNA-RNA interaction datasets indicates that 30% of detected snoRNAs interact with their host transcripts. Many snoRNA-host duplexes are located near alternatively spliced exons and display high sequence conservation suggesting a possible role in splicing regulation. The study of the model SNORD2-EIF4A2 duplex indicates that the snoRNA interaction with the host intronic sequence conceals the branch point leading to decreased inclusion of the adjacent alternative exon. Extended SNORD2 sequence containing the interacting intronic region accumulates in sequencing datasets in a cell-type-specific manner. Antisense oligonucleotides and mutations that disrupt the formation of the snoRNA-intron structure promote the splicing of the alternative exon, shifting the EIF4A2 transcript ratio away from nonsense-mediated decay. CONCLUSIONS: Many snoRNAs form RNA duplexes near alternative exons of their host transcripts, placing them in optimal positions to control host output as shown for the SNORD2-EIF4A2 model system. Overall, our study supports a more widespread role for intronic snoRNAs in the regulation of their host transcript maturation.
Assuntos
Splicing de RNA , RNA Nucleolar Pequeno , Animais , Humanos , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Íntrons , Pareamento de Bases , RNA não Traduzido/metabolismo , Mamíferos/genéticaRESUMO
The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with ß-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.