RESUMEN
BACKGROUND AND AIM: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. To improve outcomes for these patients, we need to develop new treatment strategies. Personalized cancer medicine, where patients are treated based on the characteristics of their own tumor, has gained significant interest for its promise to improve outcomes and reduce unnecessary side effects. The purpose of this study was to examine the potential utility of patient-derived colorectal cancer organoids (PDCOs) in a personalized cancer medicine setting. METHODS: Patient-derived colorectal cancer organoids were derived from tissue obtained from treatment-naïve patients undergoing surgical resection for the treatment of CRC. We examined the recapitulation of key histopathological, molecular, and phenotypic characteristics of the primary tumor. RESULTS: We created a bio-resource of PDCOs from primary and metastatic CRCs. Key histopathological features were retained in PDCOs when compared with the primary tumor. Additionally, a cohort of 12 PDCOs, and their corresponding primary tumors and normal sample, were characterized through whole exome sequencing and somatic variant calling. These PDCOs exhibited a high level of concordance in key driver mutations when compared with the primary tumor. CONCLUSIONS: Patient-derived colorectal cancer organoids recapitulate characteristics of the tissue from which they are derived and are a powerful tool for cancer research. Further research will determine their utility for predicting patient outcomes in a personalized cancer medicine setting.
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Neoplasias Colorrectales , Organoides , Estudios de Cohortes , Neoplasias Colorrectales/patología , Humanos , Organoides/patología , Medicina de PrecisiónRESUMEN
Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5' cap; the SSU then scans in the 3' direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5' UTRs. Scanning SSU have 5'-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5' start site but differ at the 3' end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.
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Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 5'/genética , Codón Iniciador/metabolismo , Codón de Terminación/metabolismo , Movimiento , Nucleótidos/genética , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Caperuzas de ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Ribosomas/químicaRESUMEN
The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαß+ CD8αα+ intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRß chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3+ regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.
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Linfocitos T CD8-positivos/citología , Regiones Determinantes de Complementariedad/química , Linfocitos Intraepiteliales/citología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Timocitos/citología , Animales , Linaje de la Célula , Cisteína/química , Ratones , Ratones Endogámicos C57BLRESUMEN
Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Macrófagos/metabolismo , Prostaglandina-E Sintasas/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/análisis , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Mapas de Interacción de Proteínas/efectos de los fármacos , Células RAW 264.7 , ARN/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.
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Regulación Fúngica de la Expresión Génica , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Sitios de Unión , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Conformación de Ácido Nucleico , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Unión Proteica , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). RESULTS: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. CONCLUSION: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.
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ADN Complementario/metabolismo , Análisis de Secuencia de ARN/métodos , Sondas de ADN/metabolismo , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/química , ARN/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Splicing introns from precursor-messenger RNA (pre-mRNA) transcripts is essential for translating functional proteins. Here, we report that the previously uncharacterized Caenorhabditis elegans protein MOG-7 acts as a pre-mRNA splicing factor. Depleting MOG-7 from the C. elegans germ line causes intron retention in most germline-expressed genes, impeding the germ cell cycle, and causing defects in nuclear morphology, germ cell identity and sterility. Despite the deleterious consequences caused by MOG-7 loss, the adult germ line can functionally recover to produce viable and fertile progeny when MOG-7 is restored. Germline recovery is dependent on a burst of apoptosis that likely clears defective germ cells, and viable gametes generated from the proliferation of germ cells in the progenitor zone. Together, these findings reveal that MOG-7 is essential for germ cell development, and that the germ line can functionally recover after a collapse in RNA splicing.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN Mensajero/metabolismoRESUMEN
Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.
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Proteínas Protozoarias/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Trypanosoma brucei brucei/genética , Animales , Ciclo Celular/fisiología , Fase G2 , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Reproducibilidad de los Resultados , Fase SRESUMEN
BACKGROUND: Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. RESULTS: We report an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, we retrieve 76% of the known cardiac developmental genes and predict 35 novel genes that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. CONCLUSIONS: Our pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.
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Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Corazón/crecimiento & desarrollo , Animales , Biología Computacional , Drosophila/genética , Drosophila/fisiología , Pruebas Genéticas , Genoma , Genómica , Interferencia de ARN , Elementos Reguladores de la Transcripción , Proteínas Ribosómicas/genéticaRESUMEN
We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.
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Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/terapia , Acinetobacter baumannii/virología , Antibacterianos/farmacología , Bacteriófagos/fisiología , Terapia de Fagos , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Animales , Cápsulas Bacterianas/virología , Proteínas del Sistema Complemento/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Femenino , Humanos , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
Trypanosoma brucei can be cultured in vitro in the mammalian bloodstream form or in the procyclic (PC) form found in the insect vector. Bloodstream trypanosomes can be cloned by limiting dilution, but PCs can only be diluted in conditioned medium, i.e., medium in which PC cells have previously been grown. It is shown here that this limitation does not apply to the most commonly used PC cell strain, Lister 427, if free radicals are removed from the medium. The reported benefit of conditioning media may arise in part from a process of hemin-catalysed depletion of peroxide ("auto-conditioning") which occurs during extended incubation at growth temperature. Scavenging free radicals by addition of pyruvate also improves PC cell viability. However, other PC cell strains such as Treu 927 require cell-conditioned media unless grown in a 5% CO2 atmosphere. Several other culture parameters that affect growth rates and dilution capability were identified.
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Medios de Cultivo , Técnicas de Cultivo/métodos , Trypanosoma brucei brucei/fisiología , Animales , Células Clonales/fisiología , Medios de Cultivo Condicionados , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
OBJECTIVE: Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown. METHODS: We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model. RESULTS: ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice. CONCLUSIONS: This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic.
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Cardiopatías Congénitas/complicaciones , Hipoglucemiantes/uso terapéutico , Síndrome Metabólico/tratamiento farmacológico , Metformina/uso terapéutico , Disfunción Ventricular Izquierda/prevención & control , Animales , Gasto Cardíaco , Metabolismo Energético , Hipoglucemiantes/administración & dosificación , Masculino , Síndrome Metabólico/complicaciones , Metformina/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Disfunción Ventricular Izquierda/tratamiento farmacológico , Disfunción Ventricular Izquierda/etiologíaRESUMEN
The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.
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Regulación de la Expresión Génica , Proteína Homeótica Nanog/genética , Células Madre Pluripotentes/fisiología , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Células Madre Embrionarias/fisiología , Ratones , Proteínas de Transporte Nucleocitoplasmático/genética , Unión Proteica , Empalme del ARNRESUMEN
Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes â¼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.
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Biosíntesis de Proteínas , Ribosomas/metabolismo , Análisis de Secuencia de ARN/métodos , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TranscriptomaRESUMEN
The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5'UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.
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Células Madre Embrionarias/fisiología , Biosíntesis de Proteínas , ARN Mensajero/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , Células-Madre Neurales/fisiología , Polirribosomas/genética , Polirribosomas/metabolismo , Empalme del ARN , Ribonucleoproteínas/genética , Análisis de Secuencia de ARNRESUMEN
Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split-adapter method including several strategies for streamlining the technique and reducing adapter dimer content.
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Sondas de ADN/metabolismo , ADN Complementario/metabolismo , Biblioteca de Genes , Técnicas Genéticas , Análisis de Secuencia de ARN , Secuencia de Bases , Cartilla de ADN/metabolismo , Reparación del ADN , ADN Circular/metabolismo , Escherichia coli/enzimología , Poliadenilación , Estabilidad del ARN , Ribonucleasa Pancreática/metabolismoRESUMEN
PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the Trypanosoma brucei genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of PUF5 did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.
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Citoplasma/metabolismo , Genoma de Protozoos/fisiología , Proteínas Protozoarias/metabolismo , ARN Protozoario/metabolismo , Proteínas de Unión al ARN/metabolismo , Trypanosoma brucei brucei/metabolismo , Citoplasma/genética , Técnicas de Silenciamiento del Gen , Proteínas Protozoarias/genética , ARN Protozoario/genética , Proteínas de Unión al ARN/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.
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Ciclo Celular/genética , Perfilación de la Expresión Génica/métodos , Transcripción Genética/genética , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Animales , Kinetoplastida/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The gelsolin gene family encodes a number of higher eukaryotic actin-binding proteins that are thought to function in the cytoplasm by severing, capping, nucleating or bundling actin filaments. Recent evidence, however, suggests that several members of the gelsolin family may have adopted unexpected nuclear functions including a role in regulating transcription. In particular, flightless I, supervillin and gelsolin itself have roles as coactivators for nuclear receptors, despite the fact that their divergence appears to predate the evolutionary appearance of nuclear receptors. Flightless I has been shown to bind both actin and the actin-related BAF53a protein, which are subunits of SWI/SNF-like chromatin remodelling complexes. The primary sequences of some actin-related proteins such as BAF53a exhibit conservation of residues that, in actin itself, are known to interact with gelsolin-related proteins. In summary, there is a growing body of evidence supporting a biological role in the nucleus for actin, Arps and actin-binding proteins and, in particular, the gelsolin family of actin-binding proteins.