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1.
Nat Commun ; 12(1): 1044, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594055

RESUMEN

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.


Asunto(s)
Bacteriófagos/genética , Microbioma Gastrointestinal/genética , Genoma Viral , Metagenoma , Codón/genética , Secuencia Conservada , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Inteínas , Intrones/genética , Sistemas de Lectura Abierta/genética , Filogenia , Empalme del ARN/genética , Transcripción Genética , /genética
2.
Nat Commun ; 12(1): 847, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558503

RESUMEN

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Asunto(s)
Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genética , Mutación/genética , Oligonucleótidos/química , Oligonucleótidos/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína C9orf72/química , Exones/genética , Glutamatos/toxicidad , Intrones/genética , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estereoisomerismo
3.
Medicine (Baltimore) ; 100(6): e24301, 2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33578525

RESUMEN

RATIONALE: Marfan syndrome (MFS) has been defined as a genetic disorder that affects various systems such as the musculoskeletal, orbital, and cardiovascular systems. Neonatal MFS is considered rare and the most severe form of MFS is characterized by rapidly progressive atrioventricular valve dysfunction, often leading to death during early childhood due to congestive heart failure. PATIENT CONCERNS: A newborn with neonatal MFS and severe cardiac involvement. He presented various severe clinical features such as arachnodactyly, camptodactyly, elbow and knee joint contracture, senile facial appearance, and deep settling with down-slanting palpebral fissure, hypoplastic ear cartilage, sagging mouth, brachycephaly, and ectopia lentis. DIAGNOSIS: Genetic analysis revealed a novel mutation at nucleotide 3964 (c.3964 + 1 G > T) in intron 32 of the fibrillin-1 gene. This mutation is identified to be in the so-called neonatal region of fibrillin-1 exon 24 to 32, as reported previously. INTERVENTIONS: The patient was managed medically for improving the low cardiac output according to severe mitral regurgitation and aortic regurgitation. Afterload reduction, full sedation, and use of diuretic were attempted to improve the oliguria and heart failure. OUTCOMES: Despite the medical management, aortic regurgitation, mitral regurgitation, pulmonary hypertension, and cardiac contractility got worse. Surgical treatment is essential to prolong the patient's life, however, considerations for the grave progression of the disease make families decide to continue palliative care instead of surgical treatment. A few months after birth, he presented with rapidly progressive aortic regurgitation, mitral regurgitation, and congestive heart failure leading to death. CONCLUSIONS: This review demonstrated the prominent characteristics of neonatal MFS mutations, it would be helpful for the recognition of novel neonatal MFS variants and valuable for the understanding of the genotype-phenotype correlations and using the plans for managements and counseling in neonatal MFS.


Asunto(s)
Anomalías Congénitas/genética , Fibrilina-1/genética , Intrones/genética , Síndrome de Marfan/genética , Anomalías Cardiovasculares/complicaciones , Sistema Cardiovascular/patología , Anomalías Congénitas/etiología , Exones/genética , Resultado Fatal , Fibrilinas/genética , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Recién Nacido , Masculino , Mutación , Oliguria/tratamiento farmacológico
4.
Nat Commun ; 12(1): 727, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33526779

RESUMEN

Alternative splicing (AS) is a fundamental step in eukaryotic mRNA biogenesis. Here, we develop an efficient and reproducible pipeline for the discovery of genetic variants that affect AS (splicing QTLs, sQTLs). We use it to analyze the GTEx dataset, generating a comprehensive catalog of sQTLs in the human genome. Downstream analysis of this catalog provides insight into the mechanisms underlying splicing regulation. We report that a core set of sQTLs is shared across multiple tissues. sQTLs often target the global splicing pattern of genes, rather than individual splicing events. Many also affect the expression of the same or other genes, uncovering regulatory loci that act through different mechanisms. sQTLs tend to be located in post-transcriptionally spliced introns, which would function as hotspots for splicing regulation. While many variants affect splicing patterns by altering the sequence of splice sites, many more modify the binding sites of RNA-binding proteins. Genetic variants affecting splicing can have a stronger phenotypic impact than those affecting gene expression.


Asunto(s)
Empalme Alternativo , Genoma Humano/genética , Sitios de Carácter Cuantitativo , Sitios de Empalme de ARN/genética , Sitios de Unión/genética , Conjuntos de Datos como Asunto , Estudio de Asociación del Genoma Completo , Humanos , Intrones/genética , Mutación , Proteínas de Unión al ARN/metabolismo , RNA-Seq , Secuenciación Completa del Genoma
5.
Nat Commun ; 12(1): 529, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33483494

RESUMEN

Aberrant splicing is a major cause of rare diseases.  However, its prediction from genome sequence alone remains in most cases inconclusive. Recently, RNA sequencing has proven to be an effective complementary avenue to detect aberrant splicing. Here, we develop FRASER, an algorithm to detect aberrant splicing from RNA sequencing data. Unlike existing methods, FRASER captures not only alternative splicing but also intron retention events. This typically doubles the number of detected aberrant events and identified a pathogenic intron retention in MCOLN1 causing mucolipidosis. FRASER automatically controls for latent confounders, which are widespread and affect sensitivity substantially. Moreover, FRASER is based on a count distribution and multiple testing correction, thus reducing the number of calls by two orders of magnitude over commonly applied z score cutoffs, with a minor loss of sensitivity. Applying FRASER to rare disease diagnostics is demonstrated by reprioritizing a pathogenic aberrant exon truncation in TAZ from a published dataset. FRASER is easy to use and freely available.


Asunto(s)
Algoritmos , Empalme Alternativo , Biología Computacional/métodos , RNA-Seq/métodos , Análisis de Secuencia de ARN/métodos , Internet , Intrones/genética , Programas Informáticos
6.
Nat Commun ; 12(1): 73, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397987

RESUMEN

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.


Asunto(s)
Epigénesis Genética , Exones/genética , Mamíferos/genética , Fase Paquiteno/genética , ARN Interferente Pequeño/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilación , Animales , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Histonas/metabolismo , Intrones/genética , Masculino , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Testículo/metabolismo , Transcripción Genética
7.
Nat Commun ; 12(1): 448, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469008

RESUMEN

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.


Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Queratinocitos/fisiología , Repitelización/genética , Células Madre/fisiología , Factores de Transcripción/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Intrones/genética , Poliadenilación/genética , Cultivo Primario de Células , Factores de Transcripción/genética
8.
Nat Commun ; 12(1): 236, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431896

RESUMEN

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)n repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.


Asunto(s)
Drosophila/genética , ARN/genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Células HeLa , Humanos , Intrones/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Fenotipo , Fosforilación , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Temperatura
9.
Gene ; 766: 145146, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32941952

RESUMEN

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer's disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer's disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.


Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Hipoxia de la Célula/genética , ARN Mensajero/genética , Proteínas tau/genética , Empalme Alternativo/genética , Encéfalo/metabolismo , Línea Celular Tumoral , Humanos , Intrones/genética , Precursores del ARN/genética , Transcripción Genética/genética
10.
Gene ; 767: 145186, 2021 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-32998045

RESUMEN

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.


Asunto(s)
Quimera/genética , Euplotes/genética , Feromonas/genética , Secuencia de Aminoácidos/genética , Secuencia de Bases/genética , Cilióforos/genética , ADN/genética , Reordenamiento Génico/genética , Intrones/genética , ARN/genética , Empalme del ARN/genética
11.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33330934

RESUMEN

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Asunto(s)
Empalme Alternativo/genética , Aminoaciltransferasas/genética , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Pliegue del ARN , Precursores del ARN/genética , ARN Mensajero/genética , Células A549 , Secuencia de Bases , Línea Celular Tumoral , Secuencia Conservada , Exones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Intrones/genética , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleótidos/genética , Oligonucleótidos Antisentido/genética , Especificidad de Órganos , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Elongación de la Transcripción Genética
12.
PLoS One ; 15(12): e0243919, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33326441

RESUMEN

Common variants in the hepatic lipase (LIPC) gene have been shown to be associated with plasma lipid levels; however, the distribution and functional features of rare and regulatory LIPC variants contributing to the extreme lipid phenotypes are not well known. This study was aimed to catalogue LIPC variants by resequencing the entire LIPC gene in 95 non-Hispanic Whites (NHWs) and 95 African blacks (ABs) with extreme HDL-C levels followed by in silico functional analyses. A total of 412 variants, including 43 novel variants were identified; 56 were unique to NHWs and 234 were unique to ABs. Seventy-eight variants in NHWs and 89 variants in ABs were present either in high HDL-C group or low HDL-C group. Two non-synonymous variants (p.S289F, p.T405M), found in NHWs with high HDL-C group were predicted to have damaging effect on LIPC protein by SIFT, MT2 and PP2. We also found several non-coding variants that possibly reside in the circRNA and lncRNA binding sites and may have regulatory potential, as identified in rSNPbase and RegulomeDB databases. Our results shed light on the regulatory nature of rare and non-coding LIPC variants as well as suggest their important contributions in affecting the extreme HDL-C phenotypes.


Asunto(s)
HDL-Colesterol/sangre , LDL-Colesterol/sangre , Lipasa/genética , Afroamericanos , Alelos , Sitios de Unión/genética , Colesterol/sangre , Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/genética , HDL-Colesterol/genética , LDL-Colesterol/genética , Grupo de Ascendencia Continental Europea , Femenino , Genotipo , Humanos , Intrones/genética , Lipasa/ultraestructura , Metabolismo de los Lípidos/genética , Lípidos/sangre , Lípidos/genética , Masculino , Polimorfismo de Nucleótido Simple , Unión Proteica/genética , Conformación Proteica , ARN Circular/sangre , ARN Circular/genética , ARN Largo no Codificante/genética , Triglicéridos/sangre , Triglicéridos/genética
13.
Development ; 147(23)2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33323375

RESUMEN

The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1r ΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1r ΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.


Asunto(s)
Desarrollo Embrionario/genética , Elementos de Facilitación Genéticos/genética , Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Sistema Nervioso Central/crecimiento & desarrollo , Embrión de Mamíferos , Intrones/genética , Ratones , Microglía/metabolismo , Tejido Parenquimatoso/crecimiento & desarrollo , Tejido Parenquimatoso/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos
14.
BMC Evol Biol ; 20(1): 164, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33308147

RESUMEN

BACKGROUND: Eukaryotic protein-coding genes consist of exons and introns. Exon-intron borders are conserved between species and thus their changes might be observed only on quite long evolutionary distances. One of the rarest types of change, in which intron relocates over a short distance, is called "intron sliding", but the reality of this event has been debated for a long time. The main idea of a search for intron sliding is to use the most accurate genome annotation and genome sequence, as well as high-quality transcriptome data. We applied them in a search for sliding introns in mammals in order to widen knowledge about the presence or absence of such phenomena in this group. RESULTS: We didn't find any significant evidence of intron sliding in the primate group (human, chimpanzee, rhesus macaque, crab-eating macaque, green monkey, marmoset). Only one possible intron sliding event supported by a set of high quality transcriptomes was observed between EIF1AX human and sheep gene orthologs. Also, we checked a list of previously observed intron sliding events in mammals and showed that most likely they are artifacts of genome annotations and are not shown in subsequent annotation versions as well as are not supported by transcriptomic data. CONCLUSIONS: We assume that intron sliding is indeed a very rare evolutionary event if it exists at all. Every case of intron sliding needs a lot of supportive data for detection and confirmation.


Asunto(s)
Evolución Molecular , Intrones/genética , Mamíferos/genética , Animales , Exones/genética , Humanos , Primates/genética , Reproducibilidad de los Resultados , Ovinos/genética , Incertidumbre
15.
Proc Natl Acad Sci U S A ; 117(52): 33404-33413, 2020 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-33376219

RESUMEN

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.


Asunto(s)
Sondas de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación de Ácido Nucleico , ARN/metabolismo , Análisis de la Célula Individual , Animales , Sistemas CRISPR-Cas/genética , Expresión Génica , Humanos , Intrones/genética , Células K562 , Riñón/citología , Ratones , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células THP-1 , Factores de Tiempo
16.
BMC Bioinformatics ; 21(1): 478, 2020 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-33099301

RESUMEN

BACKGROUND: Introns have been shown to be spliced in a defined order, and this order influences both alternative splicing regulation and splicing fidelity, but previous studies have only considered neighbouring introns. The detailed intron splicing order remains unknown. RESULTS: In this work, a method was developed that can calculate the intron splicing orders of all introns in each transcript. A simulation study showed that this method can accurately calculate intron splicing orders. I further applied this method to real S. pombe, fruit fly, Arabidopsis thaliana, and human sequencing datasets and found that intron splicing orders change from gene to gene and that humans contain more not in-order spliced transcripts than S. pombe, fruit fly and Arabidopsis thaliana. In addition, I reconfirmed that the first introns in humans are spliced slower than those in S. pombe, fruit fly, and Arabidopsis thaliana genome-widely. Both the calculated most likely orders and the method developed here are available on the web. CONCLUSIONS: A novel computational method was developed to calculate the intron splicing orders and applied the method to real sequencing datasets. I obtained intron splicing orders for hundreds or thousands of genes in four organisms. I found humans contain more number of not in-order spliced transcripts.


Asunto(s)
Arabidopsis/genética , Biología Computacional/métodos , Drosophila melanogaster/genética , Intrones/genética , Empalme del ARN/genética , Schizosaccharomyces/genética , Empalme Alternativo , Animales , Secuencia de Bases , Humanos
17.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33007253

RESUMEN

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Asunto(s)
Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Empalmosomas/metabolismo , Animales , Catálisis , Células HeLa , Humanos , Intrones/genética , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Unión Proteica , Estabilidad Proteica , ARN/química , ARN/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Factores de Tiempo
18.
Nat Commun ; 11(1): 5060, 2020 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-33033246

RESUMEN

Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.


Asunto(s)
Sistemas CRISPR-Cas/genética , Neoplasias/genética , Fusión de Oncogenes/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/genética , Doxorrubicina/uso terapéutico , Proteínas de Fusión bcr-abl/genética , Eliminación de Gen , Sitios Genéticos , Inestabilidad Genómica , Células HEK293 , Humanos , Intrones/genética , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas de Fusión Oncogénica/genética , ARN Guia/metabolismo , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto
19.
BMC Med Genet ; 21(1): 191, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004005

RESUMEN

BACKGROUND: Central nervous system (CNS) hemangioblastomas are the most frequent cause of mortality in patients with Von Hippel-Lindau (VHL) disease, an autosomal dominant genetic disease resulting from germline mutations in the VHL tumor suppressor gene, with most mutations occurring in the exons. To date, there have been no reports of CNS hemangioblastoma cases related to pathogenic variants in intron 2 of VHL, which encodes a tumor suppressor protein (i.e., pVHL) that regulates hypoxia-inducible factor proteins. CASE PRESENTATION: We report the presence of a base substitution of c.464-1G > C and c.464-2A > G in the intron 2 of VHL causing CNS hemangioblastomas in six patients with VHL from two Chinese families. The clinical information about the two pathogentic variants has been submitted to ClinVar database. The ClinVar accession for NM_000551.3(VHL):c.464-1G > C was SCV001371687. This finding may provide a new approach for diagnosing and researching VHL-associated hemangioblastomas. CONCLUSIONS: This is the first report of a pathogenic variant at intron 2 in VHL-associated hemangioblastomas. Gene sequencing showed that not only exonic but also intronic mutations can lead to the development of CNS hemangioblastomas.


Asunto(s)
Neoplasias del Sistema Nervioso Central/genética , Hemangioblastoma/genética , Mutación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/genética , Adulto , Grupo de Ascendencia Continental Asiática/genética , Secuencia de Bases , Neoplasias del Sistema Nervioso Central/diagnóstico por imagen , Neoplasias del Sistema Nervioso Central/etnología , China , Salud de la Familia , Femenino , Hemangioblastoma/diagnóstico por imagen , Hemangioblastoma/etnología , Humanos , Intrones/genética , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Linaje , Análisis de Secuencia de ADN , Enfermedad de von Hippel-Lindau/diagnóstico por imagen , Enfermedad de von Hippel-Lindau/etnología
20.
DNA Cell Biol ; 39(11): 2095-2101, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33016778

RESUMEN

Angiotensin-converting enzyme 2 (ACE2) is known as the counter-regulator of the renin-angiotensin system, it cleaves angiotensin II to render Ag 1-7, a potent vasodilator with multiple roles in cardiovascular protection. A few studies have pinpointed ACE2 polymorphisms and their relationship with heart function and hypertension in a sex-dependent manner. These observations still lack replication mostly for admixed populations. This study aimed to report minor allele frequencies of four ACE2 intron variants, rs2285666, rs2048683, rs2106809, and rs4240157, derived from previous research using the GSA, v1.0, microarray in 1231 hypertensive and nonhypertensive patients. Logistic and multiple linear regression models were developed to identify potential associations with hypertension status and systolic and diastolic blood pressure (SBP and DBP). Allele frequency differences were identified for ACE2 rs2048683 and rs4240157 in populations with European ancestry and people of the Americas. Regression analyses identified a significant association of ACE2 rs2048683 and rs4240157 with SBP/DBP in males or females. Our observations confirm sex differences in ACE2 genetic associations with SBP and DBP and contribute to the collection of genetic variation in ACE2 for admixed populations.


Asunto(s)
Presión Sanguínea/genética , Hipertensión Esencial/genética , Predisposición Genética a la Enfermedad , Peptidil-Dipeptidasa A/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Grupo de Ascendencia Continental Asiática/genética , Hipertensión Esencial/patología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Adulto Joven
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