RESUMO
The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.
Assuntos
Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Fertilização/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Oócitos/fisiologia , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Splicing de RNA/genética , Splicing de RNA/fisiologia , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismoRESUMO
Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transcrição Gênica/genética , Fator de Transcrição YY1/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genoma Humano/genética , Células Hep G2 , Humanos , Células K562 , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , RNA-Seq , Transcriptoma , Fator de Transcrição YY1/genéticaRESUMO
Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.
Assuntos
Proteínas Argonautas/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Interferente Pequeno/metabolismo , Espermatogênese , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Pareamento de Bases , Células Cultivadas , Proteína Semelhante a ELAV 1/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.
Assuntos
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Azoospermia/genética , Mutação , Animais , Azoospermia/metabolismo , Cromatina/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Histonas/metabolismo , Humanos , Íntrons , Masculino , Camundongos , Linhagem , Protaminas/metabolismo , Proteólise , Espermatogênese , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Cell replacement therapy represents a promising approach for treating neurodegenerative diseases. Contrary to the common addition strategy to generate new neurons from glia by overexpressing a lineage-specific transcription factor(s), a recent study introduced a subtraction strategy by depleting a single RNA-binding protein, Ptbp1, to convert astroglia to neurons not only in vitro but also in the brain. Given its simplicity, multiple groups have attempted to validate and extend this attractive approach but have met with difficulty in lineage tracing newly induced neurons from mature astrocytes, raising the possibility of neuronal leakage as an alternative explanation for apparent astrocyte-to-neuron conversion. This review focuses on the debate over this critical issue. Importantly, multiple lines of evidence suggest that Ptbp1 depletion can convert a selective subpopulation of glial cells into neurons and, via this and other mechanisms, reverse deficits in a Parkinson's disease model, emphasizing the importance of future efforts in exploring this therapeutic strategy.
Assuntos
Neurônios , Doença de Parkinson , Humanos , Neurônios/fisiologia , Neuroglia , Encéfalo , Astrócitos/fisiologiaRESUMO
Kinetochore assembly on centromeres is central for chromosome segregation, and defects in this process cause mitotic errors and aneuploidy. Besides the well-established protein network, emerging evidence suggests the involvement of regulatory RNA in kinetochore assembly; however, it has remained elusive about the identity of such RNA, let alone its mechanism of action in this critical process. Here, we report CCTT, a previously uncharacterized long non-coding RNA (lncRNA) transcribed from the arm of human chromosome 17, which plays a vital role in kinetochore assembly. We show that CCTT highly localizes to all centromeres via the formation of RNA-DNA triplex and specifically interacts with CENP-C to help engage this blueprint protein in centromeres, and consequently, CCTT loss triggers extensive mitotic errors and aneuploidy. These findings uncover a non-centromere-derived lncRNA that recruits CENP-C to centromeres and shed critical lights on the function of centromeric DNA sequences as anchor points for kinetochore assembly.
Assuntos
RNA Longo não Codificante , Humanos , Aneuploidia , Proteína Centromérica A/metabolismo , DNA , Cinetocoros/metabolismo , RNA Longo não Codificante/genética , CentrômeroRESUMO
Neurodegenerative diseases, characterized by progressive neural loss, have been some of the most challenging medical problems in aging societies. Treatment strategies such as symptom management have little impact on disease progression, while intervention with specific disease mechanisms may only slow down disease progression. One therapeutic strategy that has the potential to reverse the disease phenotype is to replenish neurons and rebuild the pathway lost to degeneration. Although it is generally believed that the central nervous system has lost the capability to regenerate, increasing evidence indicates that the brain is more plastic than previously thought, containing perhaps the biggest repertoire of cells with latent neurogenic programs in the body. This review focuses on key advances in generating new neurons through in situ neuronal reprogramming, which is tied to fundamental questions regarding adult neurogenesis, cell source, and mechanisms for neuronal reprogramming, as well as the ability of new neurons to integrate into the existing circuitry.
Assuntos
Doenças Neurodegenerativas , Neurônios , Encéfalo , Humanos , Doenças Neurodegenerativas/metabolismo , Neurogênese/genética , Neurônios/metabolismoRESUMO
MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here, we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1-mediated translational stimulation in the mitochondria and repression in the cytoplasm.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismo , Biossíntese de Proteínas , Animais , Proteínas Argonautas/metabolismo , Linhagem Celular , Camundongos , Mioblastos/citologia , Miócitos Cardíacos/citologiaRESUMO
Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/patologia , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas Nucleares/genética , Fosfotransferases/genética , Splicing de RNA , RNA de Transferência/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Fator de Especificidade de Clivagem e Poliadenilação/genética , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteínas Nucleares/metabolismo , Linhagem , Fosfotransferases/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
RNAP II is frequently paused near gene promoters in mammals, and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR-splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes.
Assuntos
Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA não Traduzido/metabolismo , Ribonucleoproteínas/metabolismo , Ativação Transcricional , Animais , Elementos Facilitadores Genéticos , Técnicas de Silenciamento de Genes , Camundongos , Proteínas Nucleares/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina , Elongação da Transcrição Genética , Iniciação da Transcrição GenéticaRESUMO
The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , MicroRNAs/genética , Neurônios/citologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Regulação para Baixo , Humanos , Camundongos , MicroRNAs/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Splicing de RNA , SinapsesRESUMO
The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Aciltransferases , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Humanos , Lisofosfolipídeos/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Tamanho do Órgão , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Soro/química , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The utilization of reduced plant height genes Rht-B1b and Rht-D1b, encoding homeologous DELLA proteins, led to the wheat Green Revolution (GR). However, the specific functions of GR genes in yield determination and the underlying regulatory mechanisms remained unknown. Here, we validated that Rht-B1b, as a representative of GR genes, affects plant architecture and yield component traits. Upregulation of Rht-B1b reduced plant height, leaf size and grain weight, but increased tiller number, tiller angle, spike number per unit area, and grain number per spike. Dynamic investigations showed that Rht-B1b increased spike number by improving tillering initiation rather than outgrowth, and enhanced grain number by promoting floret fertility. Rht-B1b reduced plant height by reducing cell size in the internodes, and reduced grain size or weight by decreasing cell number in the pericarp. Transcriptome analyses uncovered that Rht-B1b regulates many homologs of previously reported key genes for given traits and several putative integrators for different traits. These findings specify the pleiotropic functions of Rht-B1b in improving yield and provide new insights into the regulatory mechanisms underlying plant morphogenesis and yield formation.
Assuntos
Genes de Plantas , Triticum , Alelos , Fenótipo , Grão Comestível/metabolismo , Desenvolvimento Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2. Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson's disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.
Assuntos
Astrócitos/citologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Substância Negra/citologia , Substância Negra/fisiologia , Animais , Axônios/fisiologia , Dopamina/biossíntese , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Neostriado/citologia , Neostriado/fisiologia , Vias Neurais , Neurogênese , Doença de Parkinson/metabolismo , Fenótipo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/deficiência , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Substância Negra/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/genética , Masculino , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por SubstratoRESUMO
Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.
Assuntos
Sequência de Bases/genética , Síndromes Mielodisplásicas/genética , Fatores de Processamento de RNA/genética , Pontos de Checagem do Ciclo Celular/genética , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/genética , Fosfoproteínas/genética , Splicing de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genéticaRESUMO
Mammalian genomes are extensively transcribed, which produces a large number of both coding and non-coding transcripts. Various RNAs are physically associated with chromatin, through being either retained in cis at their site of transcription or recruited in trans to other genomic regions. Driven by recent technological innovations for detecting chromatin-associated RNAs, diverse roles are being revealed for these RNAs and associated RNA-binding proteins (RBPs) in gene regulation and genome function. Such functions include locus-specific roles in gene activation and silencing, as well as emerging roles in higher-order genome organization, such as involvement in long-range enhancer-promoter interactions, transcription hubs, heterochromatin, nuclear bodies and phase transitions.