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RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.
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Proteínas de Ligação a RNA , RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteoma/genética , FenótipoRESUMO
Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.
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RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.
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Regulação da Expressão Gênica , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Bases , Sítios de Ligação , Células HEK293 , Humanos , RNA/química , Ribonucleoproteínas/classificaçãoRESUMO
The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A)+ RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.
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Neoplasias da Mama/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Transcriptoma , Neoplasias da Mama/patologia , Reparo do DNA , Feminino , Redes Reguladoras de Genes , Humanos , Poliadenilação , Splicing de RNA , RNA Mensageiro , Radiação Ionizante , Células Tumorais CultivadasRESUMO
RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.
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Ribossomos/metabolismo , Células HEK293 , Humanos , Fases de Leitura Aberta , Biossíntese de Proteínas/genética , Ribossomos/genética , TranscriptomaRESUMO
Motivation: RNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized. Results: We developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3'UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP. Availability and implementation: SSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/. Supplementary information: Supplementary data are available at Bioinformatics online.
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Motivos de Nucleotídeos , Proteínas de Ligação a RNA/química , RNA , Sítios de Ligação , Biologia Computacional , Análise de Sequência de RNA , SoftwareRESUMO
RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.
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Antígenos de Superfície/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Antígenos de Superfície/química , Antígenos de Superfície/fisiologia , Sítios de Ligação , Biologia Computacional , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica , Células HEK293 , Humanos , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , SoftwareRESUMO
Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1(-/-) mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.
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Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Criança , Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Reagentes de Ligações Cruzadas , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Família Multigênica , Mutação , Ovário/metabolismo , Ovário/patologia , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Especificidade por SubstratoRESUMO
Pairs of RNA molecules transcribed from partially or entirely complementary loci are called cis-natural antisense transcripts (cis-NATs), and they play key roles in the regulation of gene expression in many organisms. A promising experimental tool for profiling sense and antisense transcription is strand-specific RNA sequencing (ssRNA-seq). To identify cis-NATs using ssRNA-seq, we developed a new computational method based on a model comparison framework that incorporates the inherent variable efficiency of generating perfectly strand-specific libraries. Applying the method to new ssRNA-seq data from whole-root and cell-type-specific Arabidopsis libraries confirmed most of the known cis-NAT pairs and identified 918 additional cis-NAT pairs. Newly identified cis-NAT pairs are supported by polyadenylation data, alternative splicing patterns, and RT-PCR validation. We found 209 cis-NAT pairs that have opposite expression levels in neighboring cell types, implying cell-type-specific roles for cis-NATs. By integrating a genome-wide epigenetic profile of Arabidopsis, we identified a unique chromatin signature of cis-NATs, suggesting a connection between cis-NAT transcription and chromatin modification in plants. An analysis of small-RNA sequencing data showed that â¼4% of cis-NAT pairs produce putative cis-NAT-induced siRNAs. Taken together, our data and analyses illustrate the potential for multifaceted regulatory roles of plant cis-NATs.
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Arabidopsis/genética , Biologia Computacional/métodos , RNA Antissenso/genética , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Análise de Sequência de RNA , Transcrição Gênica , Processamento Alternativo , Arabidopsis/metabolismo , Cromatina/genética , Interpretação Estatística de Dados , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Poliadenilação , Interferência de RNA , RNA Antissenso/análise , RNA de Plantas/análise , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes.
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Algoritmos , Mapeamento de Interação de Proteínas/métodos , Proteínas de Ligação a RNA/genética , RNA/genética , RNA/metabolismo , Análise de Sequência de RNA/métodos , Integração de SistemasRESUMO
Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.
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Perfilação da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Família Multigênica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , RNA/genética , Homologia de Sequência de Aminoácidos , TranscriptomaRESUMO
Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRa signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRa signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.
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Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by Nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). NMD activity varies across transcripts and cellular contexts via poorly understood mechanisms. Here, by leveraging human genetic datasets, we uncover that the amino acid preceding the PTC dramatically affects NMD activity in human cells. We find that glycine codons in particular support high levels of NMD and are enriched before PTCs but depleted before normal termination codons (NTCs). Gly-PTC enrichment is most pronounced in human genes that tolerate loss-of-function variants. This suggests a strong biological impact for Gly-PTC in ensuring robust elimination of potentially toxic truncated proteins from non-essential genes. Biochemical assays revealed that the peptide release rate during translation termination is highly dependent on the identity of the amino acid preceding the stop codon. This release rate is the most critical feature determining NMD activity across our massively parallel reporter assays. Together, we conclude that NMD activity is significantly modulated by the "window of opportunity" offered by translation termination kinetics. Integrating the window of opportunity model with the existing framework of NMD would enable more accurate nonsense variant interpretation in the clinic.
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Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.
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All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.
Assuntos
Regulação da Expressão Gênica , Herpesvirus Humano 8/genética , MicroRNAs/genética , RNA Viral/genética , Homologia de Sequência do Ácido Nucleico , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Transformação Celular Viral/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Viral/metabolismo , Especificidade por SubstratoRESUMO
The human adrenal gland consists of concentrically organized, functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from 2 individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized 2 APMs in a 72-year-old woman. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.
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HuR emerged as a posttranscriptional regulator of mRNAs involved in cellular control, stress, and immunity but its role in governing such responses remains elusive. In this study, we assessed HuR's role in the staged progression of thymic T cell differentiation by means of its genetic ablation. Mice with an early deletion of HuR in thymocytes possess enlarged thymi but display a substantial loss of peripheral T cells. We show that this discordant phenotype related to specific defects in thymic cellular processes, which demonstrated HuR's involvement in: 1) intrinsic checkpoint signals suppressing the cell cycle of immature thymocyte progenitors, 2) TCR and antigenic signals promoting the activation and positive selection of mature thymocytes, 3) antigenic and death-receptor signals promoting thymocyte deletion, and 4) chemokine signals driving the egress of postselection thymocytes to the periphery. The cellular consequences of HuR's dysfunction were underlined by the aberrant expression of selective cell cycle regulators, TCR, and death-receptor signaling components. Our studies reveal the signal-dependent context of HuR's cellular activities in thymocytes and its importance in the generation of a physiological T cell pool.
Assuntos
Antígenos de Superfície/fisiologia , Proteínas de Ligação a RNA/fisiologia , Linfócitos T/citologia , Timo/imunologia , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Quimiocinas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Camundongos , Receptores de Morte Celular , Células-Tronco/citologia , Timo/citologiaRESUMO
RNA sequencing (RNA-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total RNA is ribosomal RNA (rRNA). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger RNA (mRNA) and long noncoding RNAs (lncRNA), it is essential to minimize the rRNA being sequenced. Although commercial methods exist to deplete rRNA from total RNA samples before sequencing, they are expensive and require specific amounts of input RNA, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rRNA depletion protocol using RNase H and DNA oligonucleotides complementary to human rRNA transcripts. This protocol includes guidelines for DNA oligo preparation, RNA:DNA hybridization, RNase H cleavage and RNA cleanup, and benchmarking of rRNA depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rRNA depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input RNA amounts. © 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rRNA transcripts from human total RNA Support Protocol: Preparation of the rRNA depletion DNA oligonucleotide pool.
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RNA Ribossômico , RNA-Seq , Transcriptoma , Análise Custo-Benefício , Humanos , RNA Mensageiro , RNA Ribossômico/genéticaRESUMO
Upon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3' UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries.
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Regiões 3' não Traduzidas/genética , Proteína Semelhante a ELAV 1/metabolismo , Interferons/metabolismo , RNA Mensageiro/metabolismo , HumanosRESUMO
Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.