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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2794-2802, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472297

RESUMO

Estrogen receptor (esr) mediates the effects of estrogen on the expression of related genes, thereby regulating the growth and reproduction of mammals. To investigate the effect of retrotransposon insertion polymorphism (RIP) of the porcine esr gene on porcine growth performance, retrotransposon insertion polymorphism of the esr gene were predicted by comparative genomics and bioinformatics, and PCR was used to verify the insertion polymorphisms in different porcine breeds. Finally, the correlation analysis between the genotypes and performance of Large White pigs was conducted. The results showed that four retrotransposon polymorphic sites were identified in the esr1 and esr2 genes, which are esr1-SINE- RIP1 located in intron 2 of the esr1 gene, esr1-LINE-RIP2 and RIP3-esr1- SINE located in intron 5 of the gene, and esr2-LINE-RIP located in intron 1 of the esr2 gene, respectively. Among them, insertion of a 287 bp of SINE into intron 2 of the esr1 gene significantly affected (P<0.05) the live back fat thickness and 100 kg body weight back fat thickness of Large White pigs. Moreover, the live back fat thickness and back fat thickness at 100 kg body weight of homozygous with insertion (SINE+/+) was significantly greater than that of heterozygous with insertion (SINE+/-) and homozygous without insertion (SINE-/-). Therefore, esr1-SINE-RIP1 could be used as a molecular marker to assist the selection of deposition traits in Large White pigs.


Assuntos
Polimorfismo Genético , Retroelementos , Animais , Genótipo , Íntrons/genética , Fenótipo , Polimorfismo Genético/genética , Retroelementos/genética , Suínos/genética
2.
Commun Biol ; 4(1): 1034, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465887

RESUMO

COVID-19 has caused numerous infections with diverse clinical symptoms. To identify human genetic variants contributing to the clinical development of COVID-19, we genotyped 1457 (598/859 with severe/mild symptoms) and sequenced 1141 (severe/mild: 474/667) patients of Chinese ancestry. We further incorporated 1401 genotyped and 948 sequenced ancestry-matched population controls, and tested genome-wide association on 1072 severe cases versus 3875 mild or population controls, followed by trans-ethnic meta-analysis with summary statistics of 3199 hospitalized cases and 897,488 population controls from the COVID-19 Host Genetics Initiative. We identified three significant signals outside the well-established 3p21.31 locus: an intronic variant in FOXP4-AS1 (rs1853837, odds ratio OR = 1.28, P = 2.51 × 10-10, allele frequencies in Chinese/European AF = 0.345/0.105), a frameshift insertion in ABO (rs8176719, OR = 1.19, P = 8.98 × 10-9, AF = 0.422/0.395) and a Chinese-specific intronic variant in MEF2B (rs74490654, OR = 8.73, P = 1.22 × 10-8, AF = 0.004/0). These findings highlight an important role of the adaptive immunity and the ABO blood-group system in protection from developing severe COVID-19.


Assuntos
COVID-19/etnologia , COVID-19/genética , Grupos Étnicos/genética , Estudo de Associação Genômica Ampla , Predisposição Genética para Doença/genética , Humanos , Íntrons/genética , Polimorfismo de Nucleotídeo Único
3.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
4.
Nat Commun ; 12(1): 4910, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389706

RESUMO

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.


Assuntos
Íntrons/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Humanos , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Processamento U2AF/genética
5.
Nat Commun ; 12(1): 4908, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389711

RESUMO

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.


Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
6.
Nat Commun ; 12(1): 4825, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376658

RESUMO

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.


Assuntos
Encéfalo/metabolismo , Éxons/genética , Íntrons/genética , Sequenciamento por Nanoporos/métodos , RNA Circular/genética , Análise de Sequência de RNA/métodos , Animais , Expressão Gênica , Humanos , Masculino , Camundongos da Linhagem 129 , Isoformas de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
BMC Genomics ; 22(1): 593, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34348644

RESUMO

BACKGROUND: The mutation of insulin-like growth factor 2 (IGF2 mutation) that a single-nucleotide substitution (G→A) in the third intron of IGF2 abrogates the interaction with zinc finger BED-type containing 6 (ZBED6) and leads to increased muscle mass in pigs. IGF2 mutation knock-in (IGF2 KI) and ZBED6 knockout (ZBED6 KO) lead to changes in IGF2 expression and increase muscle mass in mice and pigs. Long noncoding RNAs (lncRNAs) may participate in numerous biological processes, including skeletal muscle development. However, the role of the ZBED6-lncRNA axis in skeletal muscle development is poorly characterized. RESULTS: In this study, we assembled transcriptomes using RNA-seq data published in previous studies by our group and identified 11,408 known lncRNAs and 2269 potential lncRNAs in seven tissues, heart, longissimus dorsi, gastrocnemius muscle, liver, spleen, lung and kidney, of ZBED6 KO (lean mass model) and WT Bama pigs. ZBED6 affected the expression of 1570 lncRNAs (differentially expressed lncRNAs [DE-lncRNAs]; log2-fold change ≥ 1, nominal p-value ≤ 0.05) in the seven examined tissues. The expressed lncRNAs (FPKM > 0.1) exhibited tissue-specific patterns in WT pigs. Specifically, 3410 lncRNAs were expressed exclusively in only one tissue. Potential functions of lncRNAs were indirectly predicted by searching their target cis- and trans-regulated protein-coding genes. LncRNAs with tissue-specific expression influence numerous genes related to tissue functions. Weighted gene coexpression network analysis (WGCNA) of 1570 DE-lncRNAs between WT and ZBED6 KO pigs was used to define the following six lncRNA modules specific to different tissues: skeletal muscle, heart, lung, spleen, kidney and liver modules. Furthermore, by conjoint analysis of longissimus dorsi data (tissue-specific expression, muscle module and DE-lncRNAs) and ChIP-PCR revealed NONSUSG002145.1 (adjusted p-values = 0.044), which is coexpressed with the IGF2 gene and binding with ZBED6, may play important roles in ZBED6 KO pig skeletal muscle development. CONCLUSIONS: These findings indicate that the identified lncRNAs may play essential roles in tissue function and regulate the mechanism of ZBED6 action in skeletal muscle development in pigs. To our knowledge, this is the first study describing lncRNAs in ZBED6 KO pigs. These results may open new research directions leading to a better understanding of the global functions of ZBED6 and of lncRNA functions in skeletal muscle development in pigs.


Assuntos
RNA Longo não Codificante , Animais , Íntrons , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Suínos/genética , Transcriptoma
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(8): 803-806, 2021 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-34365630

RESUMO

OBJECTIVE: To explore the molecular mechanism of a case where RhD genotyping did not match serological results. METHODS: The serological results of 8 members from two generations of this family were analyzed. And according to Mendelian law of inheritance, RhD genotyping, zygotic type determination and gene sequencing were performed for the family members. RESULTS: The proband and one of her cousins have the same RhD alleles, both of them have a 336-1G>A intron variant RhD allele and a complete RhD deletion allele. The variant alleles are inherited from two of their parents with blood relationship, while the complete-deleted alleles come from the other. 336-1G>A means that the last base G of the second intron of the RhD gene is mutated to A, which leads to a negative RhD serology and a positive genotype in the proband. CONCLUSION: There was a rare 336-1G> A intron variant gene (RhD * 01N.25) in this family, which was a recessive gene relative to the RhD gene and resulted in RhD phenotype negative.


Assuntos
Sistema do Grupo Sanguíneo Rh-Hr , Alelos , Feminino , Genótipo , Humanos , Íntrons/genética , Linhagem , Fenótipo , Sistema do Grupo Sanguíneo Rh-Hr/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-34281122

RESUMO

A novel CYP11A1: c.1236 + 5G > A was identified, expanding the mutation spectrum of the congenital adrenal insufficiency with 46,XY sex reversal. In a now 17-year-old girl delivered full-term (G2P2, parents unrelated), adrenal failure was diagnosed in the first year of life based on clinical picture of acute adrenal crisis with vomiting, dehydration, weight loss, hypotension, and electrolyte disturbances. At the time, hormonal tests revealed primary adrenocortical insufficiency and steroid profiles showed lack of products of steroidogenesis, and since then the patient has been treated with substitution doses of hydrocortisone and fludrocortisone. At the age of 14, considering the absence of puberty symptoms, extended diagnostic tests revealed elevated LH levels (26.5 mIU/mL) with pre-puberty FSH levels (4.9 mIU/mL), low estradiol (28 pmol/L), testosterone (<2.5 ng/mL), and extremely high levels of ACTH (4961 pg/mL). A cytogenetic study revealed a 46 XY karyotype. A molecular examination confirmed the missense mutation and a novel splice-site mutation of CYP11A1 gene. Compound heterozygosity for the CYP11A1 gene with a known pathogenic variant in one allele and a novel splice site mutation in the second allele is most probably responsible for congenital adrenal insufficiency with 46,XY sex reversal. We discuss the necessity of cytogenetic test in the case of early onset of adrenal failure in the absence of steroidogenesis metabolites in the steroid profile.


Assuntos
Insuficiência Adrenal , Enzima de Clivagem da Cadeia Lateral do Colesterol , Transtorno 46,XY do Desenvolvimento Sexual/genética , Adolescente , Insuficiência Adrenal/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Feminino , Humanos , Hidrocortisona , Íntrons , Mutação
10.
Plant Cell Rep ; 40(9): 1735-1749, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34308490

RESUMO

KEY MESSAGE: Overexpression of the naturally occurring intron-retained (IR) forms of radish RsMYB1 and RsTT8 transcripts in Arabidopsis causes a substantial increase in anthocyanin accumulation. The production of anthocyanins in plants is tightly controlled by the MYB-bHLH-WD40 (MBW) complex. In this study, analysis of four radish (Raphanus sativus L.) inbred lines with different colored taproots revealed that regulatory genes of anthocyanin biosynthesis, RsMYB1 and RsTT8, produce three transcripts, one completely spliced and two intron retention (IR1 and IR2) forms. Transcripts RsMYB1-IR1 and RsMYB1-IR2 retained the 1st (380 nt) and 2nd (149 nt) introns, respectively; RsTT8-IR1 retained the 4th intron (113 nt); RsTT8-IR2 retained both the 3rd (128 nt) and 4th introns. Levels of most IR forms were substantially low in radish samples, but the RsTT8-IR2 level was higher than RsTT8 in red skin/red flesh (RsRf) root. Since all IR forms contained a stop codon within the intron, they were predicted to encode truncated proteins with defective interaction domains, resulting in the inability to form the MBW complex in vivo. However, tobacco leaves transiently co-expressing RsMYB1-IRs and RsTT8-IRs showed substantially higher anthocyanin accumulation than those co-expressing their spliced forms. Consistently, co-expression of constructs encoding truncated proteins with spliced or IR forms of their interaction partner in tobacco leaves did not result in anthocyanin accumulation. Compared with RsMYB1, the overexpression of RsMYB1-IRs in Arabidopsis pap1 mutant increased anthocyanin accumulation by > sevenfold and upregulated the expression of Arabidopsis flavonoid biosynthesis genes including AtTT8. Our results suggest that the stable co-expression of RsMYB1-IRs in fruit trees and vegetable crops could be used to increase their anthocyanin contents.


Assuntos
Antocianinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Raphanus/genética , Processamento Alternativo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Íntrons , Pigmentação/genética , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Tabaco/genética
11.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 46(6): 658-665, 2021 Jun 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34275936

RESUMO

X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene and is the most common form of hereditary rickets. The treatment is more complicated compared with the general rickets. A family were admitted to the Department of Endocrinology, Hainan General Hospital in 2018. The proband was a 3-year-6-month-old female, Han nationality. She was admitted to hospitalization for bilateral knee valgus and walking instability. The patient's parents were not in consanguineous marrige, and there was no similar medical history in the family. The patient presented with "O" leg, bracelet sign, chicken breast, and low blood phosphorus. Typical change of rickets also appeared in her X-ray examination. The DNAs of the peripheral blood were extracted from the patient and her parents. All coding exons and flanking regions of PHEX gene in the patient were amplified by PCR, and the mutant sites of the family members were testified by a generation sequencing. A heterozygous variation (c.1482+5G>C) affecting splicing outcome was detected at the splicing region of intron 13 of PHEX gene in the patient. The variation has been included in the human gene mutation database (HGMD). No variation was found in the proband's parents, the PHEX gene in the patient was a de novo variation. Our research provided reference for the future genetic counseling for this patient and enriched the research data on the relationship between genotype and clinical manifestations.


Assuntos
Raquitismo Hipofosfatêmico Familiar , Éxons/genética , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Humanos , Lactente , Íntrons , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética
12.
J Genet ; 1002021.
Artigo em Inglês | MEDLINE | ID: mdl-34282739

RESUMO

The preproinsulin gene encodes a precursor protein of insulin, which is the most important hormone for lowering blood glucose levels and promoting the synthesis of glycogen, fat and protein. To explore the correlation between polymorphisms in the preproinsulin gene and growth traits in grass carp, the preproinsulin gene sequence, measuring a total of 5708 bp, was identified in the grass carp genome. The sequence includes a promoter, two introns and three exons, and encodes a 108-aa protein. A total of three SNPs were identified, including SNP1 (g.-2661C>G) in the promoter and SNP2 (g.1305G>C) and SNP3 (g.1682G>A) in intron 2. The correlation between SNPs and growth traits in grass carp was analysed by a general linear model (GLM). The results indicated that no genotype in each single SNP, SNP1 with SNP2, or SNP1 with SNP3 was related to rapid growth and low fatness, respectively. While eight genotypes of SNP1, SNP2 and SNP3 were combined into six types of effective diplotypes, the H5 diplotype was significantly superior to the other diplotypes (P<0.05) concerning body weight, body length, body height and body width, and its fatness was lower than those of the other diplotypes, except for H6 diplotype. This result indicated that the H5 diplotype of the preproinsulin gene in grass carp may be a candidate molecular marker for selecting fast-growing and low-fatness grass carp.


Assuntos
Carpas/genética , Insulina/genética , Regiões Promotoras Genéticas/genética , Precursores de Proteínas/genética , Animais , Sequência de Bases , Éxons/genética , Genótipo , Íntrons/genética , Polimorfismo de Nucleotídeo Único/genética
13.
Nat Commun ; 12(1): 4198, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234117

RESUMO

Our study describes breast cancer risk loci using a cross-ancestry GWAS approach. We first identify variants that are associated with breast cancer at P < 0.05 from African ancestry GWAS meta-analysis (9241 cases and 10193 controls), then meta-analyze with European ancestry GWAS data (122977 cases and 105974 controls) from the Breast Cancer Association Consortium. The approach identifies four loci for overall breast cancer risk [1p13.3, 5q31.1, 15q24 (two independent signals), and 15q26.3] and two loci for estrogen receptor-negative disease (1q41 and 7q11.23) at genome-wide significance. Four of the index single nucleotide polymorphisms (SNPs) lie within introns of genes (KCNK2, C5orf56, SCAMP2, and SIN3A) and the other index SNPs are located close to GSTM4, AMPD2, CASTOR2, and RP11-168G16.2. Here we present risk loci with consistent direction of associations in African and European descendants. The study suggests that replication across multiple ancestry populations can help improve the understanding of breast cancer genetics and identify causal variants.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Neoplasias da Mama/genética , Grupo com Ancestrais do Continente Europeu/genética , Predisposição Genética para Doença , Locos de Características Quantitativas , Feminino , Estudo de Associação Genômica Ampla , Humanos , Íntrons , Polimorfismo de Nucleotídeo Único
14.
Nat Commun ; 12(1): 4491, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301950

RESUMO

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.


Assuntos
DNA/genética , Íntrons/genética , Piranos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Compostos de Espiro/metabolismo , Spliceossomos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Humanos , Lactonas/química , Lactonas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Piranos/química , Pironas/química , Pironas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Compostos de Espiro/química , Spliceossomos/ultraestrutura
15.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281291

RESUMO

The dynamic evolution of mitochondrial gene and intron content has been reported across the angiosperms. However, a reference mitochondrial genome (mitogenome) is not available in Rubiaceae. The phylogenetic utility of mitogenome data at a species level is rarely assessed. Here, we assembled mitogenomes of six Damnacanthus indicus (Rubiaceae, Rubioideae) representing two varieties (var. indicus and var. microphyllus). The gene and intron content of D. indicus was compared with mitogenomes from representative angiosperm species and mitochondrial contigs from the other Rubiaceae species. Mitogenome structural rearrangement and sequence divergence in D. indicus were analyzed in six individuals. The size of the mitogenome in D. indicus varied from 417,661 to 419,435 bp. Comparing the number of intact mitochondrial protein-coding genes in other Gentianales taxa (38), D. indicus included 32 genes representing several losses. The intron analysis revealed a shift from cis to trans splicing of a nad1 intron (nad1i728) in D. indicus and it is a shared character with the other four Rubioideae taxa. Two distinct mitogenome structures (type A and B) were identified. Two-step direct repeat-mediated recombination was proposed to explain structural changes between type A and B mitogenomes. The five individuals from two varieties in D. indicus diverged well in the whole mitogenome-level comparison with one exception. Collectively, our study elucidated the mitogenome evolution in Rubiaceae along with D. indicus and showed the reliable phylogenetic utility of the whole mitogenome data at a species-level evolution.


Assuntos
Genoma Mitocondrial , Genoma de Planta , Rubiaceae/classificação , Rubiaceae/genética , Evolução Molecular , Rearranjo Gênico , Variação Genética , Íntrons , Filogenia , Rubiaceae/anatomia & histologia , Especificidade da Espécie , Trans-Splicing
16.
Nat Commun ; 12(1): 4507, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301951

RESUMO

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.


Assuntos
Processamento Alternativo/genética , Disautonomia Familiar/genética , Mutação , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/metabolismo , Elementos Facilitadores Genéticos/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Camundongos Transgênicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Sítios de Splice de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/metabolismo
17.
Indian J Ophthalmol ; 69(8): 2064-2070, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34304179

RESUMO

Purpose: Introns play an important role in gene regulation and expression. Single nucleotide polymorphisms (SNPs) in introns have the potential to cause disease and alter the genotype-phenotype association. Hence, this study aimed to decipher the association of SNPs in the introns of the crystallin gene in congenital cataracts. Methods: SNPs in the introns of crystallin gene family - CRYAA (rs3788059), CRYAB (rs2070894), CRYBA4 (rs2071861), and CRYBB2 (rs5752083, rs5996863) - were genotyped in 248 participants consisting of 141 congenital cataracts and 107 healthy controls by allele-specific oligonucleotide polymerase chain reaction method. Around 10% of samples for each SNPs were sequenced to confirm the genotypes. The allele, genotype, and haplotype frequency were evaluated by the SHEsis online tool. Results: Using dominant model, the "A" allele of rs3788059 was found to have an increased risk toward congenital cataract development whereas the "G" allele was found to be protective (AA + AG vs. GG; odds ratio [OR] 95% confidence interval [CI] = 3.73 [1.71, 8.15], P = 0.0009). The "A" allele of both rs2070894 (AA + AG vs. GG; OR [95% CI] = 0.49 [0.29, 0.84], P = 0.012) and rs5752083 (AA + AC vs. CC; OR [95% CI] = 0.25 [0.08, 0.76], P = 0.016) were suggested to have a protective role by the dominant model. The A-C-T haplotype (rs2071861, rs5752083, and rs5996863) was found to be a significant risk factor for the development of congenital cataract. Conclusion: Intronic SNPs in crystallin genes may play a role in the predisposition toward congenital cataract. However, the present findings need to be replicated in a large cohort with more number of samples.


Assuntos
Catarata , Cristalinas , Alelos , Catarata/genética , Cristalinas/genética , Predisposição Genética para Doença , Genótipo , Humanos , Íntrons/genética , Polimorfismo de Nucleotídeo Único
18.
Nat Commun ; 12(1): 4545, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315864

RESUMO

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5' splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5' splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5' splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3' splice sites; potentially mediating the rapid splicing of long introns.


Assuntos
Íntrons/genética , Sítios de Splice de RNA/genética , Transcrição Genética , Pareamento de Bases/genética , Sequência de Bases , Éxons/genética , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcriptoma/genética
19.
Stem Cell Res ; 54: 102438, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34214898

RESUMO

Friedreich's ataxia (FRDA) is a rare neurodegenerative disorder which is caused by triplet repeat expansion (GAA) in the first intron of FXN gene. In this present study, we generated induced pluripotent stem cells (iPSC) lines from fibroblasts of three unrelated FRDA patients using integration-free episomal vectors. All iPSC lines express the pluripotency markers such as OCT4 and SSEA4, display normal karyotypes and can differentiate into all three germ layers via in vivo teratoma formation assay.


Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Proteínas de Ligação ao Ferro , Ataxia de Friedreich/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Expansão das Repetições de Trinucleotídeos
20.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299014

RESUMO

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the PIN gene family in wheat to understand the evolution of PIN-mediated auxin transport and its role in various developmental processes and under different biotic and abiotic stress conditions. In this study, we performed genome-wide analysis of the PIN gene family in common wheat and identified 44 TaPIN genes through a homology search, further characterizing them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses led to the classification of TaPIN genes into seven different groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and three-dimensional (3D) structure were also examined using various computational approaches. Cis-elements analysis of TaPIN genes showed that TaPIN promoters consist of phytohormone, plant growth and development, and stress-related cis-elements. In addition, expression profile analysis also revealed that the expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress. Moreover, the expression patterns of TaPIN genes were verified by qRT-PCR. The qRT-PCR results also show a similar expression with slight variation. Therefore, the outcome of this study provides basic genomic information on the expression of the TaPIN gene family and will pave the way for dissecting the precise role of TaPINs in plant developmental processes and different stress conditions.


Assuntos
Ácidos Indolacéticos/metabolismo , Família Multigênica/genética , Estresse Fisiológico/genética , Triticum/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bases de Dados Genéticas , Evolução Molecular , Éxons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Ontologia Genética , Genoma de Planta , Genômica , Íntrons , Proteínas de Membrana Transportadoras/genética , Oryza/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Mapas de Interação de Proteínas , Triticum/crescimento & desenvolvimento , Triticum/metabolismo
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