RESUMO
Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.
Assuntos
Atrofia Muscular Espinal , Oligonucleotídeos Antissenso , Animais , Cromatina , Éxons , Camundongos , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Splicing de RNARESUMO
Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca2+-permeable AMPA receptor upregulation, L-type Ca2+ channel activation, enhanced spine Ca2+ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed â¼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Potenciação de Longa Duração/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Potenciais de Ação/fisiologia , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Feminino , Células HEK293 , Homeostase/fisiologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Antígeno Neuro-Oncológico Ventral , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.
Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Engenharia Genética/métodos , Engenharia de Proteínas/métodos , RNA/análise , Processamento Alternativo , Animais , Proteínas de Bactérias/metabolismo , Diferenciação Celular , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Camundongos , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , Ruminococcus , Análise de Sequência de RNA , TranscriptomaRESUMO
The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
Assuntos
Surdez/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Processamento Alternativo/genética , Animais , Linhagem Celular , Éxons , Regulação da Expressão Gênica/genética , Células HEK293 , Células Ciliadas Auditivas/fisiologia , Audição/genética , Audição/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Splicing de RNA/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição , Vorinostat/farmacologiaRESUMO
Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mamíferos/genética , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Humanos , Mamíferos/metabolismo , Isoformas de Proteínas/metabolismo , Precursores de RNA/metabolismo , Alinhamento de Sequência , Vertebrados/genética , Vertebrados/metabolismoRESUMO
Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Proteínas do Citoesqueleto/química , Humanos , Camundongos , Domínios Proteicos , Splicing de RNA , Alinhamento de Sequência , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
Assuntos
RNA Helicases DEAD-box/metabolismo , Epistasia Genética , Subunidade alfa de Receptor de Interleucina-7/genética , Splicing de RNA , RNA Helicases DEAD-box/genética , Éxons , Células HeLa , Humanos , Esclerose Múltipla/genética , Biossíntese de Proteínas , RNA Interferente Pequeno/metabolismo , Linfócitos T/imunologiaRESUMO
Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.
Assuntos
Íntrons , Metionina Adenosiltransferase/genética , Metiltransferases/metabolismo , Splicing de RNA , S-Adenosilmetionina/metabolismo , Animais , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Metionina Adenosiltransferase/química , Metilação , Metiltransferases/química , Schizosaccharomyces/metabolismoRESUMO
DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of â¼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.
Assuntos
Processamento Alternativo , Proteína do Grupo de Complementação A da Anemia de Fanconi , Humanos , Animais , Camundongos , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Reparo de DNA por Recombinação , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Células HEK293 , Éxons , Sistemas CRISPR-Cas , Reparo do DNA , Células HeLa , Dano ao DNARESUMO
Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.
Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Animais , Spliceossomos/genética , Spliceossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Íntrons/genética , Cromatina/genética , Cromatina/metabolismo , Splicing de RNA , Precursores de RNA/metabolismo , Mamíferos/metabolismoRESUMO
The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.
Assuntos
Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular , Proteína do Grupo de Complementação A da Anemia de Fanconi , Anemia de Fanconi , Splicing de RNA , Fator de Processamento U2AF , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Reparo do DNA , Endodesoxirribonucleases , Éxons , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Ligação Proteica , Precursores de RNA/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Spliceossomos/metabolismo , Spliceossomos/genética , Fator de Processamento U2AF/metabolismo , Fator de Processamento U2AF/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.
Assuntos
Éxons , Humanos , Éxons/genética , Sistemas CRISPR-Cas , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Aptidão Genética , Células HEK293 , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Sítios de Splice de RNA , Mutação , Regulação da Expressão Gênica , Processamento AlternativoRESUMO
The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.
Assuntos
Processamento Alternativo , Complexo Repressor Polycomb 2 , Animais , Camundongos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Histonas/genética , Histonas/metabolismo , Cromatina/genética , Isoformas de Proteínas/genéticaRESUMO
Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of â¼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.
Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas , Éxons , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Animais , CamundongosRESUMO
Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.
Assuntos
Processamento Alternativo , Splicing de RNA , Éxons/genética , Íntrons , RNARESUMO
4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.
Assuntos
Splicing de RNA , Pequeno RNA não Traduzido , Camundongos , Animais , Splicing de RNA/genética , Éxons/genética , Retroelementos/genética , Códon sem Sentido , Processamento AlternativoRESUMO
SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.
Assuntos
Leucemia , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , Carcinogênese/genética , Leucemia/genética , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.
Assuntos
Proteína de Ligação a Regiões Ricas em Polipirimidinas , RNA , Humanos , RNA/metabolismo , Células HeLa , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.
Assuntos
Histonas , RNA Polimerase II , Humanos , Histonas/genética , Histonas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA , Transcrição Gênica , Cromatina/genética , Processamento AlternativoRESUMO
Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.