RESUMO
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
Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many noncatalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Metalotioneína/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Cobre/toxicidade , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ligação Proteica , Clivagem do RNA , Estresse Fisiológico/efeitos dos fármacosRESUMO
Circular RNA, typically generated from backsplicing reaction, is a class of single-stranded and covalently linked RNA. Although most circular RNAs are lowly expressed, some of them are able to accumulate to high levels and even exceed their cognate mRNAs due to their longer half-lives. Once produced in the nucleus, the majority of circular RNAs are exported to the cytoplasm for their proper functions or degradation. In this review, we will summarize the biogenesis and classification of circular RNAs and highlight the recent advances in our understanding of circular RNA nuclear export and degradation.
Assuntos
Núcleo Celular/genética , Citoplasma/genética , RNA Circular/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Estabilidade de RNA , Transporte de RNARESUMO
Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases.
Assuntos
Exorribonucleases/química , Quadruplex G , RNA Mensageiro/isolamento & purificação , RNA Nuclear Pequeno/genética , RNA/isolamento & purificação , Região 3'-Flanqueadora , Células HeLa , Humanos , Lítio/farmacologia , Poliadenilação , Potássio/farmacologia , RNA/química , RNA/genética , RNA/metabolismo , Clivagem do RNA/efeitos dos fármacos , RNA Circular , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Análise de Sequência de RNARESUMO
Alternative polyadenylation (APA), which is regulated by both cis-elements and trans-factors, plays an important role in post-transcriptional regulation of eukaryotic gene expression. However, comparing to the extensively studied transcription and alternative splicing, the extent of APA divergence during evolution and the relative cis- and trans-contribution remain largely unexplored. To directly address these questions for the first time in mammals, by using deep sequencing-based methods, we measured APA divergence between C57BL/6J and SPRET/EiJ mouse strains as well as allele-specific APA pattern in their F1 hybrids. Among the 24,721 polyadenylation sites (pAs) from 7,271 genes expressing multiple pAs, we identified 3,747 pAs showing significant divergence between the two strains. After integrating the allele-specific data from F1 hybrids, we demonstrated that these events could be predominately attributed to cis-regulatory effects. Further systematic sequence analysis of the regions in proximity to cis-divergent pAs revealed that the local RNA secondary structure and a poly(U) tract in the upstream region could negatively modulate the pAs usage.
Assuntos
Processamento Alternativo , Poliadenilação , Elementos Reguladores de Transcrição , Animais , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. Hence, for most circRNAs, only a handful of sequencing reads, if any, that span the backsplicing junction are observed in standard RNA-seq libraries. It thus has become common to use the 3'-5' exonuclease ribonuclease R (RNase R) to selectively degrade linear RNAs when aiming to prove transcript circularity or biochemically enrich circRNAs. However, RNase R fails to degrade linear RNAs with structured 3' ends or internal G-quadruplex structures. To overcome these shortcomings, we describe an improved protocol for circRNA purification from total RNA that employs a poly(A) tailing step prior to RNase R digestion, which is performed in a Li+ containing buffer (rather than K+) to destabilize G-quadruplexes. This biochemical method enables higher enrichment (two- to threefold) of circRNAs to be obtained compared to standard RNase R protocols due to more efficient removal of linear RNAs. By then performing quantitative RT-PCR (RT-qPCR) or generating RNA-seq libraries, the expression of individual circRNAs can be examined or the entire set of expressed circRNAs defined using established annotation algorithms. We describe step-by-step methods for annotating circRNAs using the CIRI2 and CIRCexplorer2 algorithms. In total, this overall approach can be used to enrich for circRNAs from any total RNA sample, thereby enabling one to quickly identify and validate circRNAs of interest for functional studies.
Assuntos
Exorribonucleases , RNA Circular , RNA , RNA Mensageiro , RNA/genética , Exonucleases , DigestãoRESUMO
Caspase-8 (CASP8) involved in apoptosis plays an important role in mediating the normal regulation of cell proliferation, differentiation, inflammation, and homeostasis of multicellular organisms. Genetic polymorphisms, rs3834129 (-/CTTACT) and rs3769821 (T/C), in the promoter region of different CASP8 transcripts, were reported to be associated with genetic susceptibility of multiple cancers and non-Hodgkin's lymphoma (NHL), respectively. To investigate whether these two genetic variants, together with rs113686495 (-/CTGTCATT) which is 50 bp downstream of rs3769821, were associated with NHL in Chinese patients, we genotyped two cohorts of case and control samples from Kunming (case n = 64, control n = 133) and Shanghai (case n = 75, control n = 107). Luciferase assays were further performed to characterize the potential role of different alleles in the promoter region of the CASP8 gene. In contrast to previous studies, we found no difference regarding the genotypes and haplotypes of rs3834129, rs3769821, and rs113686495 between the case and control samples. Luciferase assays of the promoter regions harboring different alleles of these three variants also showed no difference. Our negative results gave no support for an active role for these genetic variants in conferring NHL in Chinese patients.
Assuntos
Caspase 8/genética , Linfoma não Hodgkin/genética , Proteínas de Neoplasias/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Adulto , Idoso , Estudos de Casos e Controles , Caspase 8/metabolismo , Linhagem Celular Tumoral , China , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Linfoma não Hodgkin/enzimologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismoRESUMO
Many eukaryotic protein-coding genes are able to generate exonic circular RNAs. Most of these covalently linked transcripts are expressed at low levels, but some accumulate to higher levels than their associated linear mRNAs. We highlight several methodologies that have been developed in recent years to identify and characterize these transcripts, and which have revealed an increasingly detailed view of how circular RNAs can be generated and function. It is now clear that modulation of circular RNA levels can result in a variety of molecular and physiological phenotypes, including effects on the nervous system, innate immunity, microRNAs, and many disease-relevant pathways.
Assuntos
RNA Circular/biossíntese , RNA Circular/genética , Animais , Genoma , Humanos , Sistema Imunitário/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fases de Leitura Aberta/genética , FenótipoRESUMO
Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.
Assuntos
Neoplasias Colorretais , DNA (Citosina-5-)-Metiltransferases , Regulação Neoplásica da Expressão Gênica , Isocitrato Desidrogenase , Mutação , Fator 88 de Diferenciação Mieloide , Proteínas de Neoplasias , Povo Asiático , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Feminino , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Isocitrato Desidrogenase/biossíntese , Isocitrato Desidrogenase/genética , Masculino , Fator 88 de Diferenciação Mieloide/biossíntese , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The autosomal recessive immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) is characterized by immunodeficiency, developmental delay, and facial anomalies. ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect. Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis. We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect.
Assuntos
Centrômero/genética , Instabilidade Cromossômica/genética , Face/anormalidades , Síndromes de Imunodeficiência/diagnóstico , Proteínas Repressoras/genética , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Criança , Cromossomos Humanos/genética , Metilação de DNA , Análise Mutacional de DNA , Progressão da Doença , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Mutação , Fenótipo , Doenças da Imunodeficiência PrimáriaRESUMO
BACKGROUND: Schizophrenia is recognized as a disorder of the brain and neuronal connectivity. The neural cell adhesion molecule 1 (NCAM1) gene plays a crucial role in regulating neuronal connectivity. METHODS: We conducted a two-stage association analysis on 17 NCAM1 SNPs in two independent Han Chinese schizophrenia case-control cohorts (discovery sample from Hunan Province: 986 patients and 1040 normal controls; replication sample from Yunnan Province: 564 cases and 547 healthy controls). Allele, genotype and haplotype frequencies were compared between case and control samples. Transcription factor binding site prediction and luciferase reporter assays were employed to assess the potential function of promoter SNPs. We detected developmental changes at the transcriptional level of NCAM1 during neuron differentiation in Macaca mulatta neural progenitor cells (NPC). Serum levels of NCAM1 were measured in 72 cases and 88 controls. RESULTS: A promoter variant, rs2301228, was found to be associated with schizophrenia at the allelic level and was validated in a replication cohort. Luciferase reporter assays demonstrated that risk allele rs2301228-A significantly down-regulated NCAM1 gene transcription compared to the G-allele. Concordantly, schizophrenia patients had a significantly lower level of serum NCAM1 compared to healthy donors. During the NPC neuronal differentiation, NCAM1 mRNA was significantly increased, suggesting a critical role of this gene in neural development. CONCLUSIONS: Our results provide direct evidence for NCAM1 as a susceptibility gene for schizophrenia, which offers support to a neurodevelopmental model and neuronal connectivity hypothesis in the onset of schizophrenia.
Assuntos
Antígeno CD56/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Esquizofrenia/genética , Adulto , Animais , Povo Asiático/genética , Antígeno CD56/sangue , Estudos de Casos e Controles , Células Cultivadas , China , Feminino , Haplótipos , Humanos , Macaca mulatta , Masculino , Moléculas de Adesão de Célula Nervosa/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , RNA Mensageiro/metabolismo , Esquizofrenia/sangueRESUMO
PURPOSE: Caspase 8 (CASP8) plays a critical role in the apoptotic pathway and aberrant regulation of this pathway causes many diseases including cancers. Genetic variants rs3834129 (CTTACT/-) and rs3769821 (T/C) in the promoter region of the CASP8 gene were documented to be associated with multiple solid cancers and non-Hodgkin's lymphoma (NHL), respectively, despite of some controversies. We aimed to discern potential association of these two variants and rs113686495 (CTGTCATT/-), as well as CASP8 mRNA and protein expression levels with colorectal cancer (CRC) in Han Chinese. METHODS: We genotyped CASP8 genetic variants in 305 CRC patients and 342 healthy individuals from Kunming, Southwest China. Expression levels of CASP8 mRNA and protein were quantified in paired cancerous and paracancerous normal tissues by using real-time quantitative PCR and western blot, respectively. We compared the frequencies of alleles, genotypes, and haplotypes between the cases and controls. Correlation of CASP8 mRNA and protein expression levels in paired cancerous and paracancerous normal tissues from patients with different genotypes and clinical expression were also evaluated. RESULTS: There was no association of the CASP8 genetic variants with CRC in our case-control study. The CASP8 gene mRNA expression levels in cancerous and paracancerous normal tissues were similar and there was no significant difference between subjects with different genotypes and clinical features. However, we found that CASP8 protein level was significantly lower in cancerous tissues than in paired paracancerous normal tissues. CONCLUSIONS: Our results suggest that the three CASP8 genetic variants may not be associated with CRC risk in Han Chinese from southwest China. Aberrant CASP8 protein expression may play a role in the pathogenesis of CRC.