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Classification of introns, which is crucial to understanding their evolution and splicing, has historically been binary and has resulted in the naming of major and minor introns that are spliced by their namesake spliceosome. However, a broad range of intron consensus sequences exist, leading us to here reclassify introns as minor, minor-like, hybrid, major-like, major and non-canonical introns in 263 species across six eukaryotic supergroups. Through intron orthology analysis, we discovered that minor-like introns are a transitory node for intron conversion across evolution. Despite close resemblance of their consensus sequences to minor introns, these introns possess an AG dinucleotide at the -1 and -2 position of the 5' splice site, a salient feature of major introns. Through combined analysis of CoLa-seq, CLIP-seq for major and minor spliceosome components, and RNAseq from samples in which the minor spliceosome is inhibited we found that minor-like introns are also an intermediate class from a splicing mechanism perspective. Importantly, this analysis has provided insight into the sequence elements that have evolved to make minor-like introns amenable to recognition by both minor and major spliceosome components. We hope that this revised intron classification provides a new framework to study intron evolution and splicing.
Assuntos
Evolução Molecular , Íntrons , Splicing de RNA , Spliceossomos , Íntrons/genética , Spliceossomos/genética , Humanos , Sítios de Splice de RNA , Animais , Sequência Consenso , Eucariotos/genética , Eucariotos/classificação , Sequência de BasesRESUMO
Minor intron-containing genes (MIGs) account for <2% of all human protein-coding genes and are uniquely dependent on the minor spliceosome for proper excision. Despite their low numbers, we surprisingly found a significant enrichment of MIG-encoded proteins (MIG-Ps) in protein-protein interactomes and host factors of positive-sense RNA viruses, including SARS-CoV-1, SARS-CoV-2, MERS coronavirus, and Zika virus. Similarly, we observed a significant enrichment of MIG-Ps in the interactomes and sets of host factors of negative-sense RNA viruses such as Ebola virus, influenza A virus, and the retrovirus HIV-1. We also found an enrichment of MIG-Ps in double-stranded DNA viruses such as Epstein-Barr virus, human papillomavirus, and herpes simplex viruses. In general, MIG-Ps were highly connected and placed in central positions in a network of human-host protein interactions. Moreover, MIG-Ps that interact with viral proteins were enriched with essential genes. We also provide evidence that viral proteins interact with ancestral MIGs that date back to unicellular organisms and are mainly involved in basic cellular functions such as cell cycle, cell division, and signal transduction. Our results suggest that MIG-Ps form a stable, evolutionarily conserved backbone that viruses putatively tap to invade and propagate in human host cells.
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The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of â¼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was â¼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Íntrons/genética , Neoplasias da Próstata/metabolismo , Splicing de RNA/genética , Spliceossomos/metabolismo , Transdução de Sinais , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Neoplasias de Próstata Resistentes à Castração/genéticaRESUMO
The emergence of introns was a significant evolutionary leap that is a major distinguishing feature between prokaryotic and eukaryotic genomes. While historically introns were regarded merely as the sequences that are removed to produce spliced transcripts encoding functional products, increasingly data suggests that introns play important roles in the regulation of gene expression. Here, we use an intron-centric lens to review the role of introns in eukaryotic gene expression. First, we focus on intron architecture and how it may influence mechanisms of splicing. Second, we focus on the implications of spliceosomal snRNAs and their variants on intron splicing. Finally, we discuss how the presence of introns and the need to splice them influences transcription regulation. Despite the abundance of introns in the eukaryotic genome and their emerging role regulating gene expression, a lot remains unexplored. Therefore, here we refer to introns as the "dark matter" of the eukaryotic genome and discuss some of the outstanding questions in the field.
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Splicing is a crucial regulatory node of gene expression that has been leveraged to expand the proteome from a limited number of genes. Indeed, the vast increase in intron number that accompanied vertebrate emergence might have aided the evolution of developmental and organismal complexity. Here, we review how animal models for core spliceosome components have provided insights into the role of splicing in vertebrate development, with a specific focus on neuronal, neural crest and skeletal development. To this end, we also discuss relevant spliceosomopathies, which are developmental disorders linked to mutations in spliceosome subunits. Finally, we discuss potential mechanisms that could underlie the tissue-specific phenotypes often observed upon spliceosome inhibition and identify gaps in our knowledge that, we hope, will inspire further research.
Assuntos
Proteoma , Splicing de RNA , Processamento Alternativo/genética , Animais , Íntrons , Proteoma/metabolismo , Splicing de RNA/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Vertebrados/genética , Vertebrados/metabolismoRESUMO
Minor spliceosome inhibition due to mutations in RNU4ATAC are linked to primary microcephaly. Ablation of Rnu11, which encodes a minor spliceosome snRNA, inhibits the minor spliceosome in the developing mouse pallium, causing microcephaly. There, cell cycle defects and p53-mediated apoptosis in response to DNA damage resulted in loss of radial glial cells (RGCs), underpinning microcephaly. Here, we ablated Trp53 to block cell death in Rnu11 cKO mice. We report that Trp53 ablation failed to prevent microcephaly in these double knockout (dKO) mice. We show that the transcriptome of the dKO pallium was more similar to the control compared with the Rnu11 cKO. We find aberrant minor intron splicing in minor intron-containing genes involved in cell cycle regulation, resulting in more severely impaired mitotic progression and cell cycle lengthening of RGCs in the dKO that was detected earlier than in the Rnu11 cKO. Furthermore, we discover a potential role of p53 in causing DNA damage in the developing pallium, as detection of γH2aX+ was delayed in the dKO. Thus, we postulate that microcephaly in minor spliceosome-related diseases is primarily caused by cell cycle defects.
Assuntos
Íntrons/genética , Microcefalia/genética , Splicing de RNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Ciclo Celular/genética , Morte Celular/genética , Células Ependimogliais/patologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , RNA Nuclear Pequeno/genética , Spliceossomos/genética , Transcriptoma/genéticaRESUMO
Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry-Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.
Assuntos
Processamento Alternativo , Éxons , Íntrons , Spliceossomos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatias/genética , Células Cultivadas , Ataxia Cerebelar/genética , Transtornos do Crescimento/genética , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Microcefalia/genética , Degradação do RNAm Mediada por Códon sem Sentido , Osteocondrodisplasias/genética , Polirribossomos/metabolismo , Doenças da Imunodeficiência Primária/genética , RNA Nuclear Pequeno/antagonistas & inibidores , Doenças Retinianas/genética , Fatores de Transcrição/metabolismoRESUMO
Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here, we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced owing to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase-to-metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.
Assuntos
Nanismo/genética , Extremidades/embriologia , Retardo do Crescimento Fetal/genética , Microcefalia/genética , Osteocondrodisplasias/genética , RNA Nuclear Pequeno/metabolismo , Animais , Padronização Corporal/genética , Ciclo Celular/genética , Feminino , Membro Anterior/embriologia , Membro Anterior/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/embriologia , Membro Posterior/ultraestrutura , Íntrons/genética , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , RNA Nuclear Pequeno/genética , Células-Tronco/metabolismoRESUMO
BACKGROUND: Splicing is crucial for proper gene expression, and is predominately executed by the major spliceosome. Conversely, 722 introns in 699 human minor intron-containing genes (MIGs) are spliced by the minor spliceosome. Splicing of these minor introns is disrupted in diseases caused by pathogenic variants in the minor spliceosome, ultimately leading to the aberrant expression of a subset of these MIGs. However, the effect of variants in minor introns and MIGs on diseases remains unexplored. METHODS: Variants in MIGs and associated clinical manifestations were identified using ClinVar. The HPO database was then used to curate the related symptoms and affected organ systems. RESULTS: We found pathogenic variants in 211 MIGs, which commonly resulted in intellectual disability, seizures and microcephaly. This revealed a subset of MIGs whose aberrant splicing may contribute to the pathogenesis of minor spliceosome-related diseases. Moreover, we identified 51 pathogenic variants in minor intron splice sites that reduce the splice site strength and can induce alternative splicing. CONCLUSION: These findings highlight that disrupted minor intron splicing has a broader impact on human diseases than previously appreciated. The hope is that this knowledge will aid in the development of therapeutic strategies that incorporate the minor intron splicing pathway.
Assuntos
Frequência do Gene , Doenças Genéticas Inatas/genética , Íntrons , Sítios de Splice de RNA , Processamento Alternativo , HumanosRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. While the histopathology of the different disease stages is well characterized, the cause underlying the progression, from the early drusen stage to the advanced macular degeneration stage that leads to blindness, remains unknown. Here, we show that photoreceptors (PRs) of diseased individuals display increased expression of two key glycolytic genes, suggestive of a glucose shortage during disease. Mimicking aspects of this metabolic profile in PRs of wild-type mice by activation of the mammalian target of rapamycin complex 1 (mTORC1) caused early drusen-like pathologies, as well as advanced AMD-like pathologies. Mice with activated mTORC1 in PRs also displayed other early disease features, such as a delay in photoreceptor outer segment (POS) clearance and accumulation of lipofuscin in the retinal-pigmented epithelium (RPE) and of lipoproteins at the Bruch's membrane (BrM), as well as changes in complement accumulation. Interestingly, formation of drusen-like deposits was dependent on activation of mTORC1 in cones. Both major types of advanced AMD pathologies, including geographic atrophy (GA) and neovascular pathologies, were also seen. Finally, activated mTORC1 in PRs resulted in a threefold reduction in di-docosahexaenoic acid (DHA)-containing phospholipid species. Feeding mice a DHA-enriched diet alleviated most pathologies. The data recapitulate many aspects of the human disease, suggesting that metabolic adaptations in photoreceptors could contribute to disease progression in AMD. Identifying the changes downstream of mTORC1 that lead to advanced pathologies in mouse might present new opportunities to study the role of PRs in AMD pathogenesis.
Assuntos
Envelhecimento/patologia , Macula Lutea/patologia , Degeneração Macular/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Lâmina Basilar da Corioide/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lipofuscina/metabolismo , Lipoproteínas/metabolismo , Macula Lutea/citologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
An expanded cortex is a hallmark of human neurodevelopment and endows increased cognitive capabilities. Recent work has shown that the cell cycle-related gene NDE1 is essential for proper cortical development. Patients who have mutations in NDE1 exhibit congenital microcephaly as a primary phenotype. At the cellular level, NDE1 is essential for interkinetic nuclear migration and mitosis of radial glial cells, which translates to an indispensable role in neurodevelopment. The nuclear migration function of NDE1 is well conserved across Opisthokonta. In mammals, multiple isoforms containing alternate terminal exons, which influence the functionality of NDE1, have been reported. It has been noted that the pattern of terminal exon usage mirrors patterns of cortical complexity in mammals. To provide context to these findings, here, we provide a comprehensive review of the literature regarding NDE1, its molecular biology and physiological relevance at the cellular and organismal levels. In particular, we outline the potential roles of NDE1 in progenitor cell behavior and explore the spectrum of NDE1 pathogenic variants. Moreover, we assessed the evolutionary conservation of NDE1 and interrogated whether the usage of alternative terminal exons is characteristic of species with gyrencephalic cortices. We found that gyrencephalic species are more likely to express transcripts that use the human-associated terminal exon, whereas lissencephalic species tend to express transcripts that use the mouse-associated terminal exon. Among gyrencephalic species, the human-associated terminal exon was preferentially expressed by those with a high order of gyrification. These findings underscore phylogenetic relationships between the preferential usage of NDE1 terminal exon and high-order gyrification, which provide insight into cortical evolution underlying high-order brain functions.
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Minor introns constitute <0.5% of the introns in the human genome and have remained an enigma since their discovery. These introns are removed by a distinct splicing complex, the minor spliceosome. Both are ancient, tracing back to the last eukaryotic common ancestor (LECA), which is reflected by minor intron enrichment in specific gene families, such as the mitogen activated-protein kinase kinases, voltage-gated sodium and calcium ion channels, and E2F transcription factors. Most minor introns occur as single introns in genes with predominantly major introns. Due to this organization, minor intron-containing gene (MIG) expression requires the coordinated action of two spliceosomes, which increases the probability of missplicing. Thus, one would expect loss of minor introns via purifying selection. This has resulted in complete minor intron loss in at least nine eukaryotic lineages. However, minor introns are highly conserved in land plants and metazoans, where their importance is underscored by embryonic lethality when the minor spliceosome is inactivated. Conditional inactivation of the minor spliceosome has shown that rapidly dividing progenitor cells are highly sensitive to minor spliceosome loss. Indeed, we found that MIGs were significantly enriched in a screen for genes essential for survival in 341 cycling cell lines. Here, we propose that minor introns inserted randomly into genes in LECA or earlier and were subsequently conserved in genes crucial for cycling cell survival. We hypothesize that the essentiality of MIGs allowed minor introns to endure through the unicellularity of early eukaryotic evolution. Moreover, we identified 59 MIGs that emerged after LECA, and that many of these are essential for cycling cell survival, reinforcing our essentiality model for MIG conservation. This suggests that minor intron emergence is dynamic across eukaryotic evolution, and that minor introns should not be viewed as molecular fossils. We also posit that minor intron splicing was co-opted in multicellular evolution as a regulatory switch for en masse control of MIG expression and the biological processes they regulate. Specifically, this mode of regulation could control cell proliferation and thus body size, an idea supported by domestication syndrome, wherein MIGs are enriched in common candidate animal domestication genes.
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Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here, we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.
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Axônios/metabolismo , Complexo Nuclear Basolateral da Amígdala/fisiologia , Córtex Cerebral/fisiologia , Aprendizagem , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Ratos , Análise de Sequência de RNARESUMO
BACKGROUND: Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues. RESULTS: We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns. CONCLUSION: Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu .
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Processamento Alternativo , Íntrons , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fases de Leitura Aberta/genética , Especificidade de Órgãos/genética , Especificidade de Órgãos/fisiologia , Splicing de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genéticaRESUMO
Mutation in minor spliceosome components is linked to the developmental disorder microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Here, we inactivated the minor spliceosome in the developing mouse cortex (pallium) by ablating Rnu11, which encodes the crucial minor spliceosome small nuclear RNA (snRNA) U11. Rnu11 conditional knockout mice were born with microcephaly, which was caused by the death of self-amplifying radial glial cells (RGCs), while intermediate progenitor cells and neurons were produced. RNA sequencing suggested that this cell death was mediated by upregulation of p53 (Trp53 - Mouse Genome Informatics) and DNA damage, which were both observed specifically in U11-null RGCs. Moreover, U11 loss caused elevated minor intron retention in genes regulating the cell cycle, which was consistent with fewer RGCs in S-phase and cytokinesis, alongside prolonged metaphase in RGCs. In all, we found that self-amplifying RGCs are the cell type most sensitive to loss of minor splicing. Together, these findings provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.
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Ciclo Celular/genética , Morte Celular/fisiologia , Nanismo/genética , Células Ependimogliais/metabolismo , Retardo do Crescimento Fetal/genética , Microcefalia/genética , Células-Tronco Neurais/citologia , Osteocondrodisplasias/genética , Splicing de RNA/genética , RNA Nuclear Pequeno/genética , Spliceossomos/genética , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Spliceossomos/metabolismo , Proteína Supressora de Tumor p53/biossínteseRESUMO
BACKGROUND: The retina as a model system with extensive information on genes involved in development/maintenance is of great value for investigations employing deep sequencing to capture transcriptome change over time. This in turn could enable us to find patterns in gene expression across time to reveal transition in biological processes. METHODS: We developed a bioinformatics pipeline to categorize genes based on their differential expression and their alternative splicing status across time by binning genes based on their transcriptional kinetics. Genes within same bins were then leveraged to query gene annotation databases to discover molecular programs employed by the developing retina. RESULTS: Using our pipeline on RNA-Seq data obtained from fractionated (nucleus/cytoplasm) developing retina at embryonic day (E) 16 and postnatal day (P) 0, we captured high-resolution as in the difference between the cytoplasm and the nucleus at the same developmental time. We found de novo transcription of genes whose transcripts were exclusively found in the nuclear transcriptome at P0. Further analysis showed that these genes enriched for functions that are known to be executed during postnatal development, thus showing that the P0 nuclear transcriptome is temporally ahead of that of its cytoplasm. We extended our strategy to perform temporal analysis comparing P0 data to either P21-Nrl-wildtype (WT) or P21-Nrl-knockout (KO) retinae, which predicted that the KO retina would have compromised vasculature. Indeed, histological manifestation of vasodilation has been reported at a later time point (P60). CONCLUSIONS: Thus, our approach was predictive of a phenotype before it presented histologically. Our strategy can be extended to investigating the development and/or disease progression of other tissue types.
Assuntos
Retina/metabolismo , Transcriptoma , Processamento Alternativo , Animais , Biologia Computacional , Progressão da Doença , Perfilação da Expressão Gênica , Cinética , Camundongos , Camundongos Knockout , Retina/anormalidades , Retina/embriologia , Análise de Sequência de RNA , Análise Espaço-TemporalRESUMO
In eukaryotes, gene expression requires splicing, which starts with the identification of exon-intron boundaries by the small, nuclear RNA (snRNAs) of the spliceosome, aided by associated proteins. In the mammalian genome, <1% of introns lack canonical exon-intron boundary sequences and cannot be spliced by the canonical splicing machinery. These introns are spliced by the minor spliceosome, consisting of unique snRNAs (U11, U12, U4atac, and U6atac). The importance of the minor spliceosome is underscored by the disease microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), which is caused by mutation in U4atac. Thus, it is important to understand the expression and function of the minor spliceosome and its targets in mammalian development, for which we used the mouse as our model. Here, we report enrichment of the minor snRNAs in the developing head/central nervous system (CNS) between E9.5 and E12.5, along with enrichment of these snRNAs in differentiating retinal neurons. Moreover, dynamic expression kinetics of minor intron-containing genes (MIGs) was observed across retinal development. DAVID analysis of MIGs that were cotranscriptionally upregulated embryonically revealed enrichment for RNA metabolism and cell cycle regulation. In contrast, MIGs that were cotranscriptionally upregulated postnatally revealed enrichment for protein localization/transport, vesicle-mediated transport, and calcium transport. Finally, we used U12 morpholino to inactivate the minor spliceosome in the postnatal retina, which resulted in apoptosis of differentiating retinal neurons. Taken together, our data suggest that the minor spliceosome may have distinct functions in embryonic versus postnatal development. Importantly, we show that the minor spliceosome is crucial for the survival of terminally differentiating retinal neurons.
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Neurogênese , RNA Nuclear Pequeno/metabolismo , Retina/embriologia , Retina/metabolismo , Neurônios Retinianos/fisiologia , Spliceossomos/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Eletroporação , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Análise em Microsséries , Microscopia Confocal , Microscopia de Fluorescência , Morfolinos , Neurônios Retinianos/patologiaRESUMO
In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.
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Senescência Celular/genética , Replicação do DNA/genética , Histonas/metabolismo , Neurogênese/genética , Neurônios Retinianos/fisiologia , Transcrição Gênica , Animais , Ciclina D1/metabolismo , Ciclina E/metabolismo , Histonas/genética , Camundongos , Isoformas de Proteínas/genética , RNA Mensageiro/fisiologia , Células-Tronco/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
During development, multiple cell types within a tissue often arise from a common pool of progenitor cells (PCs). PCs typically expand in number, while simultaneously producing post-mitotic cells (PMCs). This balance is partly regulated by transcription factors that are expressed within PCs, such as the basic helix-loop-helix (bHLH) gene mouse atonal homolog 7 (Math5), which is expressed in retinal PCs. Here we report that alternative splicing (AS) of Math5 serves as another layer of regulation of Math5 activity. Specifically, Math5, a single exon gene, is alternatively spliced such that the major isoform lacks the entire coding sequence. Similarly, neurogenin 3 (Ngn3), a Math5 paralog expressed in pancreatic PCs, is also alternatively spliced such that the major isoform fails to code for Ngn3 protein. The consequence of reducing the abundance of protein-coding isoforms is likely crucial, as we found that introduction of coding isoforms leads to a reduction in cycling PCs. Thus, AS can limit the number of PCs expressing key regulatory proteins that control PC expansion versus PMC production.
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Processamento Alternativo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular , Embrião de Mamíferos/metabolismo , Éxons , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismoRESUMO
Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.