RESUMO
The processing of RNA transcripts from mammalian genes occurs in proximity to their transcription. Here, we describe a phenomenon affecting thousands of genes that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts promoter choice and the expression level of the gene. We observed that evolutionary gain of internal exons is associated with gain of new transcription start sites (TSSs) nearby and increased gene expression. Inhibiting exon splicing reduced transcription from nearby promoters, and creation of new spliced exons activated transcription from cryptic promoters. The strongest effects occurred for weak promoters located proximal and upstream of efficiently spliced exons. Together, our findings support a model in which splicing recruits transcription machinery locally to influence TSS choice and identify exon gain, loss, and regulatory change as major contributors to the evolution of alternative promoters and gene expression in mammals.
Assuntos
Éxons , Regiões Promotoras Genéticas , Ativação Transcricional/genética , Células 3T3 , Animais , Evolução Molecular , Células HeLa , Humanos , Camundongos , Splicing de RNA , Sítio de Iniciação de TranscriçãoRESUMO
Alternative splicing (AS) greatly expands the coding capacities of genomes by allowing the generation of multiple mature mRNAs from a limited number of genes. Although the massive switch in AS profiles that often accompanies variations in gene expression patterns occurring during cell differentiation has been characterized for a variety of models, their causes and mechanisms remain largely unknown. Here, we integrate foundational and recent studies indicating the AS switches that govern the processes of cell fate determination. We include some distinct AS events in pluripotent cells and somatic reprogramming and discuss new progresses on alternative isoform expression in adipogenesis, myogenic differentiation and stimulation of immune cells. Finally, we cover novel insights on AS mechanisms during neuronal differentiation, paying special attention to the role of chromatin structure.
Assuntos
Processamento Alternativo , Diferenciação Celular/genética , Animais , HumanosRESUMO
Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.
Assuntos
Processamento Alternativo/fisiologia , Diferenciação Celular/fisiologia , Cromatina/genética , Epigênese Genética/fisiologia , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/fisiologia , Processamento Alternativo/genética , Animais , Azacitidina/farmacologia , Azepinas/farmacologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Metilação de DNA/efeitos dos fármacos , Primers do DNA/genética , Epigênese Genética/genética , Éxons/genética , Camundongos , Quinazolinas/farmacologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Assuntos
Processamento Alternativo/fisiologia , Elongação da Transcrição Genética/fisiologia , Processamento Alternativo/genética , Animais , Cromatina/química , Cromatina/metabolismo , Cromatina/fisiologia , Humanos , Cinética , Modelos Biológicos , Ligação Proteica/fisiologia , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologiaRESUMO
The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.
RESUMO
Long-read RNA sequencing has shed light on transcriptomic complexity, but questions remain about the functionality of downstream protein products. We introduce Biosurfer, a computational approach for comparing protein isoforms, while systematically tracking the transcriptional, splicing, and translational variations that underlie differences in the sequences of the protein products. Using Biosurfer, we analyzed the differences in 32,799 pairs of GENCODE annotated protein isoforms, finding a majority (70%) of variable N-termini are due to the alternative transcription start sites, while only 9% arise from 5' UTR alternative splicing. Biosurfer's detailed tracking of nucleotide-to-residue relationships helped reveal an uncommonly tracked source of single amino acid residue changes arising from the codon splits at junctions. For 17% of internal sequence changes, such split codon patterns lead to single residue differences, termed "ragged codons". Of variable C-termini, 72% involve splice- or intron retention-induced reading frameshifts. We found an unusual pattern of reading frame changes, in which the first frameshift is closely followed by a distinct second frameshift that restores the original frame, which we term a "snapback" frameshift. We analyzed long read RNA-seq-predicted proteome of a human cell line and found similar trends as compared to our GENCODE analysis, with the exception of a higher proportion of isoforms predicted to undergo nonsense-mediated decay. Biosurfer's comprehensive characterization of long-read RNA-seq datasets should accelerate insights of the functional role of protein isoforms, providing mechanistic explanation of the origins of the proteomic diversity driven by the alternative splicing. Biosurfer is available as a Python package at https://github.com/sheynkman-lab/biosurfer.
RESUMO
Cancer development and progression are generally associated with dysregulation of gene expression, often resulting from changes in transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network as well as an extensive promoter clone resource for future studies. Most highly connected TFs do not show a preference for binding to promoters of genes associated with either good or poor cancer prognosis, suggesting that emerging strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activator or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF estrogen receptor É (ESR1) on DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study not only broadens our knowledge of TFs involved in the cancer gene regulatory network but also provides a valuable resource for future studies, laying a foundation for potential therapeutic strategies targeting TFs in cancer.
RESUMO
Cancer development and progression are generally associated with gene dysregulation, often resulting from changes in the transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network, as well as an extensive promoter clone resource for future studies. Highly connected TFs bind to promoters of genes associated with either good or poor cancer prognosis, suggesting that strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene-targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activators or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF ESR1 in DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study broadens our knowledge of the TFs involved in cancer gene regulation and provides a valuable resource for future studies and therapeutics.
Assuntos
DNA , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias , Ligação Proteica , Fatores de Transcrição , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , DNA/metabolismo , DNA/genética , Regiões Promotoras Genéticas/genética , Oncogenes/genética , Prognóstico , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Biologia Computacional/métodosRESUMO
Transcription and splicing are intrinsically coupled. Alternative splicing of internal exons can fine-tune gene expression through a recently described phenomenon called exon-mediated activation of transcription starts (EMATS). However, the association of this phenomenon with human diseases remains unknown. Here, we develop a strategy to activate gene expression through EMATS and demonstrate its potential for treatment of genetic diseases caused by loss of expression of essential genes. We first identified a catalog of human EMATS genes and provide a list of their pathological variants. To test if EMATS can be used to activate gene expression, we constructed stable cell lines expressing a splicing reporter based on the alternative splicing of motor neuron 2 (SMN2) gene. Using small molecules and antisense oligonucleotides (ASOs) currently used for treatment of spinal muscular atrophy, we demonstrated that increase of inclusion of alternative exons can trigger an activation of gene expression up to 45-fold by enhancing transcription in EMATS-like genes. We observed the strongest effects in genes under the regulation of weak human promoters located proximal to highly included skipped exons.
Assuntos
Atrofia Muscular Espinal , Splicing de RNA , Humanos , Processamento Alternativo/genética , Éxons/genética , Regiões Promotoras Genéticas/genética , Linhagem Celular , Atrofia Muscular Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Messenger RNA isoform differences are predominantly driven by alternative first, internal, and last exons. Despite the importance of classifying exons to understand isoform structure, few tools examine isoform-specific exon usage. We recently observed that alternative transcription start sites often arise near internal exons, often creating "hybrid" first/internal exons. To systematically detect hybrid exons, we built the hybrid-internal-terminal (HIT) pipeline to classify exons depending on their isoform-specific usage. On the basis of splice junction reads in RNA sequencing data and probabilistic modeling, the HIT index identified thousands of previously misclassified hybrid first-internal and internal-last exons. Hybrid exons are enriched in long genes and genes involved in RNA splicing and have longer flanking introns and strong splice sites. Their usage varies considerably across human tissues. By developing the first method to classify exons according to isoform contexts, our findings document the occurrence of hybrid exons, a common quirk of the human transcriptome.
Assuntos
Processamento Alternativo , Transcriptoma , Sequência de Bases , Éxons , Humanos , Íntrons/genéticaRESUMO
The generation of axonal and dendritic domains is critical for brain circuitry assembly and physiology. Negative players, such as the RhoA-Rho coiled-coil-associated protein kinase (ROCK) signaling pathway, restrain axon development and polarization. Surprisingly, the genetic control of neuronal polarity has remained largely unexplored. Here, we report that, in primary cultured neurons, expression of the histone methyltransferase G9a and nuclear translocation of its major splicing isoform (G9a/E10+) peak at the time of axon formation. RNAi suppression of G9a/E10+ or pharmacological blockade of G9a constrains neuronal migration, axon initiation, and the establishment of neuronal polarity in situ and in vitro. Inhibition of G9a function upregulates RhoA-ROCK activity by increasing the expression of Lfc, a guanine nucleotide exchange factor (GEF) for RhoA. Together, these results identify G9a as a player in neuronal polarization.
Assuntos
Axônios/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Axônios/enzimologia , Movimento Celular/fisiologia , Células Cultivadas , Epigênese Genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Gravidez , Ratos , Ratos Wistar , Transdução de Sinais , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Although important information is available on the molecular mechanisms of long-term memory formation, little is known about the processes underlying memory persistence in the brain. Here, we report that persistent gene expression of CaMKIIδ isoform participates in object recognition long-lasting memory storage in mice hippocampus. We found that CaMKIIδ mRNA expression was sustained up to one week after training and paralleled memory retention. Antisense DNA infusion in the hippocampus during consolidation or even after consolidation impairs 7-day- but not 1-day-long memory, supporting a role of CaMKIIδ in memory persistence. CaMKIIδ gene expression was accompanied by long-lasting nucleosome occupancy changes at its promoter. This epigenetic mechanism is described for the first time in a memory process and offers a novel mechanism for persistent gene expression in neurons. CaMKIIδ protein is mainly present in nucleus and presynaptic terminals, suggesting a role in these subcellular compartments for memory persistence. All these results point to a key function of the sustained gene expression of this overlooked CaMKII isoform in long-lasting memories.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Neurônios/metabolismo , Animais , Medo/fisiologia , Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
Maternal malnutrition is one of the major early-life adversities affecting the development of newborn's brain and is associated with an increased risk to acquire cognitive and emotional deficiencies later in life. Studies in rodents have demonstrated that exposure to an enriched environment (EE) can reverse the negative consequences of early adversities. However, rescue of emotional disorders caused by perinatal malnutrition and the mechanisms involved has not been determined. We hypothesized that exposure to an EE may attenuate the anxiety-like disorders observed in mice subjected to perinatal protein malnutrition and that this could be mediated by epigenetic mechanisms. Male CF-1 mice were subject to perinatal protein malnutrition until weaning and then exposed to an EE for 5â¯weeks after which small RNA-seq was performed. In parallel, dark-light box and elevated plus maze tests were conducted to evaluate anxiety traits. We found that exposure to an EE reverses the anxiety-like behavior in malnourished mice. This reversal is paralleled by the expression of three miRNAs that become dysregulated by perinatal malnutrition (miR-187-3p, miR-369-3p and miR-132-3p). The predicted mRNA targets of these miRNAs are mostly related to axon guidance pathway. Accordingly, we also found that perinatal malnutrition leads to reduction in the cingulum size and altered oligodendrocyte morphology. These results suggest that EE-rescue of anxiety disorders derived from perinatal malnutrition is mediated by the modulation of miRNAs associated with the regulation of genes involved in axonal guidance.
Assuntos
Ansiedade/metabolismo , Encéfalo/metabolismo , Meio Ambiente , Regulação da Expressão Gênica , Desnutrição/metabolismo , MicroRNAs/metabolismo , Oligodendroglia/metabolismo , Animais , Ansiedade/etiologia , Ansiedade/patologia , Comportamento Animal/fisiologia , Encéfalo/patologia , Forma Celular/fisiologia , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Abrigo para Animais , Desnutrição/complicações , Desnutrição/patologia , Camundongos , MicroRNAs/genética , Oligodendroglia/patologiaRESUMO
Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs.
RESUMO
Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.
Assuntos
Processamento Alternativo , Histona-Lisina N-Metiltransferase/genética , Animais , Azepinas/farmacologia , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Éxons , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Células HeLa , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tretinoína/farmacologiaRESUMO
Sociobiology, the study of social behavior, calls for a laboratory model with specific requirements. Among the most obvious is the execution of social interactions that need to be readily observable, quantifiable and analyzable. If, in turn, one focuses on the neuroendocrinological basis of social behavior, restrictions grow even tighter. A good laboratory model should then allow easy access to its neurological and endocrine components and processes. During the last years, we have been studying the physiological foundation of social behavior on what we believe fits all the aforementioned requirements: the so called "chanchita", Cichlasoma dimerus. This Neotropical cichlid fish exhibits biparental care of the eggs and larvae and presents a hierarchical social system, established and sustained through agonistic interactions. The aim of the current article is to review new evidence on chanchita's social and reproductive behavior.
Assuntos
Ciclídeos/fisiologia , Sistema Endócrino/fisiologia , Hormônios/fisiologia , Comportamento Sexual Animal/fisiologia , Comportamento Social , Agressão/fisiologia , Animais , Feminino , Masculino , FotoperíodoRESUMO
Splicing and alternative splicing are involved in the expression of most human genes, playing key roles in differentiation, cell cycle progression, and development. Misregulation of splicing is frequently associated to disease, which imposes a better understanding of the mechanisms underlying splicing regulation. Accumulated evidence suggests that multiple trans-acting factors and cis-regulatory elements act together to determine tissue-specific splicing patterns. Besides, as splicing is often cotranscriptional, a complex picture emerges in which splicing regulation not only depends on the balance of splicing factor binding to their pre-mRNA target sites but also on transcription-associated features such as protein recruitment to the transcribing machinery and elongation kinetics. Adding more complexity to the splicing regulation network, recent evidence shows that chromatin structure is another layer of regulation that may act through various mechanisms. These span from regulation of RNA polymerase II elongation, which ultimately determines splicing decisions, to splicing factor recruitment by specific histone marks. Chromatin may not only be involved in alternative splicing regulation but in constitutive exon recognition as well. Moreover, splicing was found to be necessary for the proper 'writing' of particular chromatin signatures, giving further mechanistic support to functional interconnections between splicing, transcription and chromatin structure. These links between chromatin configuration and splicing raise the intriguing possibility of the existence of a memory for splicing patterns to be inherited through epigenetic modifications.
Assuntos
Cromatina , Splicing de RNA , Processamento Alternativo , Sequência de Bases , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Análise de Sequência de RNA , Transcrição GênicaRESUMO
Timing of breeding to an optimal season is a requirement for a successful reproductive outcome in seasonally breeding species. Photoperiodic signals are capable of modifying the reproductive behaviour and reproductive systems in several vertebrate species. The cichlid fish Cichlasoma dimerus shows highly organized breeding activities and different social status. The aim of this study is to test whether C. dimerus reproductive behaviour (male aggressive behaviour and female choice) and reproductive physiology (GnRH3 morphometric parameters, pituitary hormones content and organ-somatic indexes) are modulated by photoperiod. Before spawning, dominant pairs were isolated and kept in opposite tanks of 20 l for one week, so they could see each other but not physically interact. Afterwards, a group was exposed for four weeks to a short photoperiod (8h light:16 h dark) (short photoperiod exposed animals: SP) while another group was exposed to a long photoperiod (14 h light:10h dark) (long photoperiod exposed animals: LP). Temperature was maintained constant. Behavioural experiments showed that male aggression related to territory selection and its defence is reduced in SP males. Further, SP females were never chosen. At the brain level we demonstrated that GnRH3 neuronal optical density of staining was reduced. Finally, at the pituitary level we showed that SP males showed low levels of beta-LH, PRL and GH in the pituitary, and that SP females showed no significant differences in the pituitary content of any hormone. Taken all together these results suggest that in C. dimerus the photoperiod is a relevant environmental cue related to reproductive behaviour and physiology.