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1.
Nat Commun ; 12(1): 1214, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33619278

RESUMEN

Melanoma is the most lethal skin malignancy, driven by genetic and epigenetic alterations in the complex tumour microenvironment. While large-scale molecular profiling of melanoma has identified molecular signatures associated with melanoma progression, comprehensive systems-level modeling remains elusive. This study builds up predictive gene network models of molecular alterations in primary melanoma by integrating large-scale bulk-based multi-omic and single-cell transcriptomic data. Incorporating clinical, epigenetic, and proteomic data into these networks reveals key subnetworks, cell types, and regulators underlying melanoma progression. Tumors with high immune infiltrates are found to be associated with good prognosis, presumably due to induced CD8+ T-cell cytotoxicity, via MYO1F-mediated M1-polarization of macrophages. Seventeen key drivers of the gene subnetworks associated with poor prognosis, including the transcription factor ZNF180, are tested for their pro-tumorigenic effects in vitro. The anti-tumor effect of silencing ZNF180 is further validated using in vivo xenografts. Experimentally validated targets of ZNF180 are enriched in the ZNF180 centered network and the known pathways such as melanoma cell maintenance and immune cell infiltration. The transcriptional networks and their critical regulators provide insights into the molecular mechanisms of melanomagenesis and pave the way for developing therapeutic strategies for melanoma.


Asunto(s)
Redes Reguladoras de Genes , Melanoma/patología , Modelos Biológicos , Neoplasias Cutáneas/patología , Microambiente Tumoral , Línea Celular Tumoral , Reparación del ADN , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Interferón gamma/metabolismo , Melanoma/genética , Miosina Tipo I/metabolismo , Invasividad Neoplásica , Pronóstico , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Neoplasias Cutáneas/genética , Análisis de Supervivencia , Microambiente Tumoral/genética , Regulación hacia Arriba/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.
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
4.
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
6.
Neuron ; 109(2): 191-192, 2021 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-33476558

RESUMEN

In this issue of Neuron, Chai et al. (2021) analyze several families with neurodegeneration and marked pontocerebellar hypoplasia and microcephaly and identify recessive (bi-allelic) mutations in peptidyl-prolyl isomerase-like 1 (PPIL1) and pre-RNA-processing-17 (PPR17). PPIL1 patient mutation knockin mice develop neuronal apoptosis. Loss of either protein affects splicing predominantly involving GC-rich and short introns.


Asunto(s)
Enfermedades Cerebelosas , Microcefalia , Animales , Humanos , Ratones , Mutación/genética , Empalme del ARN/genética
7.
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
8.
Gene ; 765: 145129, 2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-32905827

RESUMEN

Hereditary spastic paraplegia (HSP) is a heterogeneous group of genetic disorders characterized by lower-limb spastic paralysis. We report on a family with three generations of autosomal dominant inheritance of HSP caused by a novel heterozygous splice-site mutation (c.303 + 2 T > C) in REEP1 that was confirmed by RFLP analysis. Carriers of the mutation, including one asymptomatic individual, showed a mild HSP phenotype with a wide range of intrafamilial variation. All symptomatic carriers had ankle contractures in addition to other classical clinical symptoms of HSP. Clinicians should suspect REEP1-related HSP in patients who show ankle contractures with other symptoms of HSP and should consider that these patients have asymptomatic carriers within their family.


Asunto(s)
Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Paraplejía Espástica Hereditaria/genética , Adulto , Familia , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación , Linaje , Fenotipo , Empalme del ARN/genética , Paraplejía Espástica Hereditaria/fisiopatología
9.
J Vis Exp ; (166)2020 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-33369604

RESUMEN

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.


Asunto(s)
Galectina 3/genética , Microesferas , Empalme del ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Núcleo Celular/metabolismo , Galectina 3/metabolismo , Células HeLa , Humanos , Péptidos/metabolismo , Precursores del ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética
10.
Nat Commun ; 11(1): 5608, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33154379

RESUMEN

The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.


Asunto(s)
Proteínas de Drosophila/genética , Intrones , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Empalmosomas/patología , Animales , Modelos Animales de Enfermedad , Drosophila , Atrofia Muscular Espinal/patología , Mutación , Fenotipo , Sitios de Empalme de ARN , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Empalmosomas/genética
11.
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
12.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2357-2360, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-33018480

RESUMEN

In the past decades an extensive mathematical literature was developed to model and analyze gene networks under both deterministic and stochastic formalisms. However, such literature is predominantly focused to deal with the modeling of transcriptional and translational regulation, but results related to post-transcriptional regulation and its connection with transcriptional regulation are poorly investigated. However, it is becoming of paramount importance the need for modeling post-transcriptional regulation via splicing especially for minor organisms or viruses.The aim of this study is to propose a first general basic modeling scheme for modeling gene expression via alternative splicing and investigating the basic deterministic and stochastic features of the pre-mRNA, mRNAs and proteins under different biological conditions.This first study showed the dynamical properties of alternative splicing, the faster kinetics of the pre-mRNA compared to the mRNA and the importance to stochastically model gene networks when considering the post-transcriptional regulation.


Asunto(s)
Precursores del ARN , Empalme del ARN , Empalme Alternativo , Redes Reguladoras de Genes , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN Mensajero/genética
13.
Nat Commun ; 11(1): 4744, 2020 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-32958768

RESUMEN

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.


Asunto(s)
Sitios de Empalme de ARN/genética , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Exones/genética , Humanos , Mutación , Neoplasias/química , Neoplasias/genética , Nucleótidos , Motivo de Reconocimiento de ARN , Empalme del ARN/genética , Factor de Empalme U2AF/química , Dedos de Zinc
14.
Int J Oral Sci ; 12(1): 22, 2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32737282

RESUMEN

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Saco Dental/citología , Displasia de la Dentina/genética , Displasia de la Dentina/fisiopatología , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Osteogénesis/genética , Empalme del ARN/genética , Estudios de Casos y Controles , Diferenciación Celular/genética , Células Cultivadas , Displasia de la Dentina/patología , Humanos , Mutación/genética
15.
Int J Hematol ; 112(6): 894-899, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32772263

RESUMEN

Diamond-Blackfan Anemia (DBA) is a congenital pure red cell aplasia caused by heterozygous variants in ribosomal protein genes. The hematological features associated with DBA are highly variable and non-hematological abnormalities are common. We report herein on an affected mother and her daughter presenting with transfusion-dependent anemia. The mother showed mild physical abnormalities and entered spontaneous remission at age 13 years. Her daughter was born with occipital meningocele. Exome sequencing of DNA from the mother revealed a heterozygous novel splice site variant (NM_001011.4:c.508-3T > G) in the Ribosomal Protein S7 gene (RPS7) inherited by the daughter. Functional analysis of the RPS7 variant expressed from a mini-gene construct revealed that the exon 7 acceptor splice site was replaced by a cryptic splice resulting in a transcript missing 64 bp of exon 7 (p.Val170Serfs*8). Our study confirms a pathogenic effect of a novel RPS7 variant in DBA associated with spontaneous remission in the mother and meningocele in her daughter, thus adding to the genotype-phenotype correlations in DBA.


Asunto(s)
Anemia de Diamond-Blackfan/genética , Aberraciones Cromosómicas , Estudios de Asociación Genética , Variación Genética/genética , Meningocele/genética , Empalme del ARN/genética , Proteínas Ribosómicas/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/etiología , Niño , Exones/genética , Femenino , Humanos , Meningocele/etiología , Relaciones Madre-Hijo , Remisión Espontánea , Análisis de Secuencia de ADN
16.
Nat Commun ; 11(1): 4140, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32811829

RESUMEN

Investigating the evolution of complex phenotypes and the underlying molecular bases of their variation is critical to understand how organisms adapt to their environment. Applying classical quantitative genetics on a segregating population derived from a Can-0xCol-0 cross, we identify the MADS-box transcription factor FLOWERING LOCUS M (FLM) as a player of the phenotypic variation in plant growth and color. We show that allelic variation at FLM modulates plant growth strategy along the leaf economics spectrum, a trade-off between resource acquisition and resource conservation, observable across thousands of plant species. Functional differences at FLM rely on a single intronic substitution, disturbing transcript splicing and leading to the accumulation of non-functional FLM transcripts. Associations between this substitution and phenotypic and climatic data across Arabidopsis natural populations, show how noncoding genetic variation at a single gene might be adaptive through pleiotropic effects.


Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Empalme del ARN/genética , Alelos , Arabidopsis/metabolismo , Evolución Molecular , Pleiotropía Genética , Variación Genética , Intrones , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Sitios de Carácter Cuantitativo/genética , Temperatura
17.
Nucleic Acids Res ; 48(16): 9250-9261, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32813009

RESUMEN

N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.


Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/genética , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , Adenosina/genética , Catálisis , Exones/genética , Humanos , Metilación , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , Empalmosomas/genética
18.
PLoS Biol ; 18(7): e3000782, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32692742

RESUMEN

Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Proteínas de Unión al Calcio/metabolismo , Desarrollo de la Planta/genética , Empalme del ARN/genética , Transcripción Genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al Calcio/genética , Núcleo Celular/metabolismo , Flores/fisiología , Histonas/metabolismo , Mutación con Pérdida de Función , Lisina/metabolismo , Metilación , Proteínas Nucleares/metabolismo , Fenotipo , Raíces de Plantas/citología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Aminoácido
19.
PLoS Genet ; 16(7): e1008944, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32730252

RESUMEN

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.


Asunto(s)
Chlamydomonas reinhardtii/genética , Intrones/genética , Procesamiento Proteico-Postraduccional/genética , Empalme del ARN/genética , Regulación de la Expresión Génica de las Plantas/genética , Microalgas/genética , Regiones Promotoras Genéticas , Transcriptoma/genética
20.
Nat Genet ; 52(9): 939-949, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32601472

RESUMEN

N6-methyladenosine (m6A) plays important roles in regulating messenger RNA processing. Despite rapid progress in this field, little is known about the genetic determinants of m6A modification and their role in common diseases. In this study, we mapped the quantitative trait loci (QTLs) of m6A peaks in 60 Yoruba (YRI) lymphoblastoid cell lines. We found that m6A QTLs are largely independent of expression and splicing QTLs and are enriched with binding sites of RNA-binding proteins, RNA structure-changing variants and transcriptional features. Joint analysis of the QTLs of m6A and related molecular traits suggests that the downstream effects of m6A are heterogeneous and context dependent. We identified proteins that mediate m6A effects on translation. Through integration with data from genome-wide association studies, we show that m6A QTLs contribute to the heritability of various immune and blood-related traits at levels comparable to splicing QTLs and roughly half of expression QTLs. By leveraging m6A QTLs in a transcriptome-wide association study framework, we identified putative risk genes of these traits.


Asunto(s)
Adenosina/análogos & derivados , ARN Mensajero/genética , Adenosina/genética , Mapeo Cromosómico/métodos , Pruebas Genéticas/métodos , Variación Genética/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Fenotipo , Sitios de Carácter Cuantitativo/genética , Carácter Cuantitativo Heredable , Empalme del ARN/genética , Transcriptoma/genética
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