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1.
Genome Res ; 29(12): 2046-2055, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31727681

RESUMEN

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.


Asunto(s)
Algoritmos , Empalme Alternativo , Bases de Datos de Ácidos Nucleicos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Péptidos , Isoformas de ARN , Humanos , Péptidos/genética , Péptidos/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de ARN/biosíntesis , Isoformas de ARN/genética , Espectrometría de Masas en Tándem
2.
RNA ; 26(10): 1320-1333, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32554554

RESUMEN

Human CD4+ T cells are often subdivided into distinct subtypes, including Th1, Th2, Th17, and Treg cells, that are thought to carry out distinct functions in the body. Typically, these T-cell subpopulations are defined by the expression of distinct gene repertoires; however, there is variability between studies regarding the methods used for isolation and the markers used to define each T-cell subtype. Therefore, how reliably studies can be compared to one another remains an open question. Moreover, previous analysis of gene expression in CD4+ T-cell subsets has largely focused on gene expression rather than alternative splicing. Here we take a meta-analysis approach, comparing eleven independent RNA-seq studies of human Th1, Th2, Th17, and/or Treg cells to determine the consistency in gene expression and splicing within each subtype across studies. We find that known master-regulators are consistently enriched in the appropriate subtype; however, cytokines and other genes often used as markers are more variable. Importantly, we also identify previously unknown transcriptomic markers that appear to consistently differentiate between subsets, including a few Treg-specific splicing patterns. Together this work highlights the heterogeneity in gene expression between samples designated as the same subtype, but also suggests additional markers that can be used to define functional groupings.


Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Expresión Génica/genética , Empalme del ARN/genética , Subgrupos de Linfocitos T/fisiología , Transcriptoma/genética , Adulto , Células Cultivadas , Citocinas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad
3.
PLoS Biol ; 15(9): e2002623, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28961236

RESUMEN

Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5.


Asunto(s)
Factor 2B Eucariótico de Iniciación/genética , Perfilación de la Expresión Génica/métodos , Intrones/genética , Biosíntesis de Proteínas/genética , Hipoxia Tumoral/genética , Empalme Alternativo/genética , Secuencia de Bases , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Sitios Genéticos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Modelos Biológicos , Motivos de Nucleótidos/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reproducibilidad de los Resultados
4.
J Biol Chem ; 292(44): 18240-18255, 2017 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-28916722

RESUMEN

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3ß phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.


Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Empalme Alternativo , Animales , Isótopos de Carbono , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/metabolismo , Técnicas de Inactivación de Genes , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas de Homeodominio/química , Ratones , Isótopos de Nitrógeno , Proteínas Nucleares/química , Nucleofosmina , Mapeo Peptídico , Fosforilación , Estabilidad Proteica , Proteómica/métodos , Proteínas de Unión al ARN/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras , Factores de Empalme Serina-Arginina/química , Especificidad por Sustrato
5.
J Immunol ; 196(9): 3768-79, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-27036912

RESUMEN

Microbial colonization of the infant gastrointestinal tract (GIT) begins at birth, is shaped by the maternal microbiota, and is profoundly altered by antibiotic treatment. Antibiotic treatment of mothers during pregnancy influences colonization of the GIT microbiota of their infants. The role of the GIT microbiota in regulating adaptive immune function against systemic viral infections during infancy remains undefined. We used a mouse model of perinatal antibiotic exposure to examine the effect of GIT microbial dysbiosis on infant CD8(+) T cell-mediated antiviral immunity. Maternal antibiotic treatment/treated (MAT) during pregnancy and lactation resulted in profound alterations in the composition of the GIT microbiota in mothers and infants. Streptococcus spp. dominated the GIT microbiota of MAT mothers, whereas Enterococcus faecalis predominated within the MAT infant GIT. MAT infant mice subsequently exhibited increased and accelerated mortality following vaccinia virus infection. Ag-specific IFN-γ-producing CD8(+) T cells were reduced in sublethally infected MAT infant mice. MAT CD8(+) T cells from uninfected infant mice also demonstrated a reduced capacity to sustain IFN-γ production following in vitro activation. We additionally determined that control infant mice became more susceptible to infection if they were born in an animal facility using stricter standards of hygiene. These data indicate that undisturbed colonization and progression of the GIT microbiota during infancy are necessary to promote robust adaptive antiviral immune responses.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Enterococcus faecalis/fisiología , Microbioma Gastrointestinal , Streptococcus/fisiología , Virus Vaccinia/inmunología , Vaccinia/microbiología , Inmunidad Adaptativa , Animales , Animales Recién Nacidos , Antibacterianos/administración & dosificación , Antibacterianos/efectos adversos , Células Cultivadas , Femenino , Interferón gamma/metabolismo , Exposición Materna/efectos adversos , Ratones , Ratones Endogámicos C57BL , Vaccinia/inmunología
6.
J Gen Virol ; 97(7): 1537-1544, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27072634

RESUMEN

GB virus C (GBV-C) is a non-pathogenic flavivirus that may play a role in modulating HIV disease. Multiple genotypes of GBV-C that have been identified to date that may differentially regulate HIV; however, the number of complete GBV-C sequences published to date is very limited. We sequenced full-length GBV-C genomes from four individuals with HIV/HCV co-infection in the United States. Intergenotypic recombination was evident in two of these individuals. Evaluation of additional full-length GBV-C genomes would facilitate the creation of full-length, replication-competent molecular clones of GBV-C to evaluate the phenotypic diversity of GBV-C genotypes and provide important molecular data on this understudied virus.


Asunto(s)
Virus GB-C/genética , Virus GB-C/aislamiento & purificación , Genoma Viral/genética , Recombinación Genética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Coinfección , Infecciones por Flaviviridae/virología , Humanos , Filogenia , Estudios Prospectivos , ARN Viral/genética , Análisis de Secuencia de ARN , Estados Unidos
7.
Nat Commun ; 14(1): 1230, 2023 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-36869033

RESUMEN

The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we assess the advantages of MAJIQ v2 compared to existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer insights into brain subregion-specific splicing regulation.


Asunto(s)
Algoritmos , Empalme del ARN , RNA-Seq , Benchmarking , Encéfalo
8.
Elife ; 112022 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-36264057

RESUMEN

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here, we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (TCR, CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax, and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together, these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.


Asunto(s)
Antígenos CD28 , Linfocitos T , Humanos , Linfocitos T/metabolismo , Antígenos CD28/metabolismo , Empalme Alternativo , Supervivencia Celular , Receptores de Antígenos de Linfocitos T/metabolismo , Apoptosis
9.
Nat Commun ; 12(1): 3353, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34099673

RESUMEN

The effects of confounding factors on gene expression analysis have been extensively studied following the introduction of high-throughput microarrays and subsequently RNA sequencing. In contrast, there is a lack of equivalent analysis and tools for RNA splicing. Here we first assess the effect of confounders on both expression and splicing quantifications in two large public RNA-Seq datasets (TARGET, ENCODE). We show quantification of splicing variations are affected at least as much as those of gene expression, revealing unwanted sources of variations in both datasets. Next, we develop MOCCASIN, a method to correct the effect of both known and unknown confounders on RNA splicing quantification and demonstrate MOCCASIN's effectiveness on both synthetic and real data. Code, synthetic and corrected datasets are all made available as resources.


Asunto(s)
Algoritmos , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Empalme del ARN , Bases de Datos Genéticas , Humanos , Células K562 , RNA-Seq/métodos , Reproducibilidad de los Resultados , Programas Informáticos
10.
Dev Cell ; 43(3): 318-331.e5, 2017 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-29107558

RESUMEN

Alternative splicing contributes to gene expression dynamics in many tissues, yet its role in auditory development remains unclear. We performed whole-exome sequencing in individuals with sensorineural hearing loss (SNHL) and identified pathogenic mutations in Epithelial Splicing-Regulatory Protein 1 (ESRP1). Patient-derived induced pluripotent stem cells showed alternative splicing defects that were restored upon repair of an ESRP1 mutant allele. To determine how ESRP1 mutations cause hearing loss, we evaluated Esrp1-/- mouse embryos and uncovered alterations in cochlear morphogenesis, auditory hair cell differentiation, and cell fate specification. Transcriptome analysis revealed impaired expression and splicing of genes with essential roles in cochlea development and auditory function. Aberrant splicing of Fgfr2 blocked stria vascularis formation due to erroneous ligand usage, which was corrected by reducing Fgf9 gene dosage. These findings implicate mutations in ESRP1 as a cause of SNHL and demonstrate the complex interplay between alternative splicing, inner ear development, and auditory function.


Asunto(s)
Empalme Alternativo/genética , Cóclea/embriología , Pérdida Auditiva/genética , Mutación/genética , Proteínas de Unión al ARN/genética , Animales , Diferenciación Celular/genética , Cóclea/metabolismo , Ratones Noqueados
11.
Biosens Bioelectron ; 62: 320-4, 2014 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-25038536

RESUMEN

Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 µM arsenite with a linear range up to 100 µM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.


Asunto(s)
Arsénico/análisis , Técnicas Biosensibles/métodos , Shewanella/genética , Shewanella/metabolismo , Arsénico/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Técnicas Electroquímicas , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/análisis , Genes Bacterianos , Ingeniería Genética , Hierro/metabolismo , Oxidación-Reducción , Regiones Promotoras Genéticas
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