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1.
BMC Evol Biol ; 19(1): 162, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375061

RESUMO

BACKGROUND: Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS: We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS: Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.


Assuntos
Sequência Conservada , Diplomonadida/genética , Íntrons/genética , Parabasalídeos/genética , Filogenia , Spliceossomos/genética , Regiões 5' não Traduzidas/genética , Pareamento de Bases/genética , Sequência de Bases , Genoma , Conformação de Ácido Nucleico , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Proteínas Ribossômicas/genética
2.
Nat Commun ; 10(1): 2983, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278301

RESUMO

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/genética , Humanos , Microscopia Intravital , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Processamento de RNA/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade
3.
Nat Med ; 25(6): 911-919, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160820

RESUMO

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.


Assuntos
Doenças Raras/genética , Ceramidase Ácida/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Variação Genética , Humanos , Masculino , Modelos Genéticos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Canais de Potássio/genética , RNA/sangue , RNA/genética , Processamento de RNA/genética , Doenças Raras/sangue , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
4.
Mol Biol (Mosk) ; 53(3): 524-528, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184618

RESUMO

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.


Assuntos
Proteínas/análise , Proteínas/química , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Sequências Repetitivas de Aminoácidos/genética , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas/genética , Proteoma/análise , Proteoma/química , Proteoma/genética , Processamento de RNA/genética
5.
Nat Commun ; 10(1): 2569, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189880

RESUMO

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.


Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Mutação Silenciosa/genética , Conjuntos de Dados como Assunto , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dobramento de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
6.
Medicine (Baltimore) ; 98(21): e15743, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31124956

RESUMO

BACKGROUND: Gene mutations with important prognostic role have been identified in patients with myelodysplastic syndrome (MDS). We performed a meta-analysis to investigate the effects of RNA splicing machinery gene mutations on prognosis of MDS patients. METHODS: We searched English database including PubMed, Embase, Cochrane Library for literatures published within recent 10 years on the effect of RNA splicing machinery genes in MDS. Revman version 5.2 software was used for all the statistical processing. We calculated risk ratio and 95% confidence interval (CI) of continuous variables, and find hazard ratio (HR) and 95% CI of time-to-event data. RESULTS: We included 19 studies enrolling 4320 patients. There is a significant superior overall survival (OS) in splicing factor 3b, subunit 1 (SF3B1)-mutation group compared to unmutated group (HR = 0.58, 95% CI: 0.5-0.67, P < .00001); OS decreased significantly in serine/arginine-rich splicing factor 2/ U2 auxiliary factor protein 1 (SRSF2/U2AF1) mutation group compared to unmutated group, (HR = 1.62, 95% CI: 1.34-1.97, P < .00001 and HR = 1.61, 95% CI: 1.35-1.9, P < .00001, respectively). In terms of leukemia-free survival (LFS), the group with SF3B1 mutation had better outcome than unmutated group, HR = 0.63 (95% CI: 0.53-0.75, P < .00001). Other RNA splicing gene mutation group showed significant poor LFS than unmutated groups, (HR = 1.89, 95% CI: 1.6-2.23, P < .00001; HR = 2.77, 95% CI: 2.24-3.44, P < .00001; HR = 1.48, 95% CI: 1.08-2.03, P < .00001; for SRSF2, U2AF1, and zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 [ZRSR2], respectively). As for subgroup of low- or intermediate-1-IPSS risk MDS, SRSF2, and U2AF1 mutations were related to poor OS. (HR = 1.83, 95% CI: 1.43-2.35, P < .00001; HR = 2.11, 95% CI: 1.59-2.79, P < .00001 for SRSF2 and U2AF1, respectively). SRSF2 and U2AF1 mutations were strongly associated with male patients. SF3B1 mutation was strongly associated with disease staging. CONCLUSION: This meta-analysis indicates a positive effect of SF3B1 and an adverse prognostic effect of SRSF2, U2AF1, and ZRSR2 mutations in patients with MDS. Mutations of RNA splicing genes have important effects on the prognosis of MDS.


Assuntos
Síndromes Mielodisplásicas/genética , Processamento de RNA/genética , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Índice de Gravidade de Doença , Fatores Sexuais , Fator de Processamento U2AF/genética , Análise de Sobrevida
7.
Plant Cell Physiol ; 60(8): 1734-1746, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31076755

RESUMO

Pentatricopeptide repeat (PPR) proteins play crucial roles in intron splicing, which is important for RNA maturation. Identification of novel PPR protein with the function of intron splicing would help to understand the RNA splicing mechanism. In this study, we identified the maize empty pericarp602 (emp602) mutants, the mature kernels of which showed empty pericarp phenotype. We cloned the Emp602 gene from emp602 mutants and revealed that Emp602 encodes a mitochondrial-localized P-type PPR protein. We further revealed that Emp602 is specific for the cis-splicing of mitochondrial Nad4 intron 1 and intron 3, and mutation of Emp602 led to the loss of mature Nad4 transcripts. The loss of function of Emp602 nearly damaged the assembly and accumulation of complex I and arrested mitochondria formation, which arrested the seed development. The failed assembly of complex I triggers significant upregulation of Aox expression in emp602 mutants. Transcriptome analysis showed that the expression of mitochondrial-related genes, e.g. the genes associated with mitochondrial inner membrane presequence translocase complex and electron carrier activity, were extensively upregulated in emp602 mutant. These results demonstrate that EMP602 functions in the splicing of Nad4 intron 1 and intron 3, and the loss of function of Emp602 arrested maize seed development by disrupting the mitochondria complex I assembly.


Assuntos
Sementes/metabolismo , Zea mays/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Íntrons/genética , Íntrons/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Processamento de RNA/genética , Processamento de RNA/fisiologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/genética , Zea mays/crescimento & desenvolvimento
8.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086020

RESUMO

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Processamento de RNA/genética , Spodoptera/genética , Processamento Alternativo/genética , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Feminino , Masculino , Ligação Proteica
9.
Biochim Biophys Acta Gene Regul Mech ; 1862(6): 634-642, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31042550

RESUMO

Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5'ss marked by GU dinucleotides defines the 5'ss as well as facilitates 3'ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5'ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5'ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.


Assuntos
Sítios de Splice de RNA , Processamento de RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Processamento Alternativo , Éxons/genética , Humanos , Íntrons/genética , Mutação , Processamento de RNA/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor
10.
BMC Bioinformatics ; 20(1): 231, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068132

RESUMO

BACKGROUND: In eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited. RESULTS: Here, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript. CONCLUSIONS: SpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip.


Assuntos
Processamento Alternativo/genética , Éxons/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/genética
11.
Nat Commun ; 10(1): 1338, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902979

RESUMO

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.


Assuntos
Alelos , Variação Genética , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Regiões 3' não Traduzidas/genética , Motivos de Aminoácidos , Sequência de Bases , Biologia Computacional , Simulação por Computador , Doença/genética , Predisposição Genética para Doença , Células Hep G2 , Humanos , Células K562 , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Locos de Características Quantitativas/genética , RNA Helicases/metabolismo , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transativadores/metabolismo
12.
Jpn J Clin Oncol ; 49(5): 477-480, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30882153

RESUMO

The proband was a 62-year-old man with ureter cancer. He had a history of metachronous colorectal and gastric cancer. Immunohistochemical staining showed the absence of both MSH2 and MSH6 proteins in the ureter cancer and other available cancer tissue specimens. Genetic testing was conducted to identify the causative genes of hereditary gastrointestinal cancer syndromes including mismatch repair genes. We detected a germline variant, c.2635-3delC, within the splice acceptor site of exon 16, in the MSH2 gene. To investigate whether this variant affected splicing of the gene, RNA sequencing was performed using blood samples. We observed a substantial amount of the transcripts that lacked proper splicing of intron 15 in the indexed case, whereas, a very low amount of such aberrant transcripts was detected in the controls, strongly indicating an association between the variant and splicing defect. These results indicate that MSH2 c.2635-3delC affects normal splicing and might be a cause of Lynch syndrome.


Assuntos
Pareamento de Bases/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Predisposição Genética para Doença , Íntrons/genética , Proteína 2 Homóloga a MutS/genética , Processamento de RNA/genética , Deleção de Sequência , Adulto , Sequência de Bases , Simulação por Computador , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
13.
Int J Hematol ; 109(5): 603-611, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30850927

RESUMO

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.


Assuntos
Subunidade gama Comum de Receptores de Interleucina , Mutação , Sítios de Splice de RNA , Processamento de RNA , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Adolescente , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Processamento de RNA/genética , Processamento de RNA/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologia
14.
Biomed Res Int ; 2019: 2756516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834258

RESUMO

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, like O. sativa, Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs. By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, we have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, our method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica/métodos , Processamento de RNA/genética , RNA/genética , Animais , Arabidopsis/genética , Genoma/genética , Humanos , Camundongos , MicroRNAs/genética , Anotação de Sequência Molecular , Oryza/genética , RNA/isolamento & purificação
15.
Oncogene ; 38(21): 4142-4153, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30705407

RESUMO

Novel therapeutic strategies for non-small-cell lung cancer (NSCLC) are urgently needed. RNA splicing, orchestrated by the spliceosome, is deregulated in many forms of cancer, including NSCLC. Here, we performed high-throughput screening with a small interfering RNA library targeting all annotated human spliceosome proteins to identify cancer-selective lethal targets in the RNA splicing machinery. Silencing of several spliceosome proteins reduced cell viability in a panel of NSCLC cell lines, but not in non-malignant lung fibroblasts and epithelial cells. Interestingly, the cancer-selective lethal target set comprised all seven Sm proteins that, together with small nuclear RNA, form the core structure of most spliceosome subunits. Interfering with Sm protein expression induced apoptosis in NSCLC cells, but not in non-malignant cells. In silico analysis revealed that Sm proteins are frequently upregulated in NSCLC. For several Sm proteins, increased expression showed a positive correlation with disease severity. Together, our results suggest that the Sm proteins represent particularly useful novel targets for selective treatment of NSCLC.


Assuntos
Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Spliceossomos/genética , Células A549 , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/patologia , Fibroblastos/patologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Processamento de RNA/genética , Regulação para Cima/genética
16.
Methods Mol Biol ; 1935: 175-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30758827

RESUMO

Single-cell RNA-seq (scRNA-seq) provides a comprehensive measurement of stochasticity in transcription, but the limitations of the technology have prevented its application to dissect variability in RNA processing events such as splicing. In this chapter, we review the challenges in splicing isoform quantification in scRNA-seq data and discuss BRIE (Bayesian regression for isoform estimation), a recently proposed Bayesian hierarchical model which resolves these problems by learning an informative prior distribution from sequence features. We illustrate the usage of BRIE with a case study on 130 mouse cells during gastrulation.


Assuntos
Isoformas de Proteínas/genética , Processamento de RNA/genética , RNA Citoplasmático Pequeno/genética , Análise de Sequência de RNA/métodos , Animais , Teorema de Bayes , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , RNA/genética , Análise de Célula Única/métodos , Software
17.
Nat Commun ; 10(1): 521, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705266

RESUMO

Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.


Assuntos
Éxons/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Processamento de RNA/genética
18.
Int J Mol Sci ; 20(3)2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759747

RESUMO

TSG101 (Tumor susceptibility 101) gene and its aberrantly spliced isoform, termed TSG101∆154-1054, are tightly linked to tumorigenesis in various cancers. The aberrant TSG101∆154-1054 mRNA is generated from cancer-specific re-splicing of mature TSG101 mRNA. The TSG101∆154-1054 protein protects the full-length TSG101 protein from ubiquitin-mediated degradation, implicating TSG101∆154-1054 protein in the progression of cancer. Here, we confirmed that the presence of TSG101∆154-1054 mRNA indeed caused an accumulation of the TSG101 protein in biopsies of human nasopharyngeal carcinoma (NPC), which was recapitulated by the overexpression of TSG101∆154-1054 in the NPC cell line TW01. We demonstrate the potential function of the TSG101∆154-1054 protein in the malignancy of human NPC with scratch-wound healing and transwell invasion assays. By increasing the stability of the TSG101 protein, TSG101∆154-1054 specifically enhanced TSG101-mediated TW01 cell migration and invasion, suggesting the involvement in NPC metastasis in vivo. This finding sheds light on the functional significance of TSG101∆154-1054 generation via re-splicing of TSG101 mRNA in NPC metastasis and hints at its potential importance as a therapeutic target.


Assuntos
Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Metástase Neoplásica/genética , Processamento de RNA/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Adulto , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica
19.
Nat Genet ; 51(4): 755-763, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804562

RESUMO

Exome analysis of patients with a likely monogenic disease does not identify a causal variant in over half of cases. Splice-disrupting mutations make up the second largest class of known disease-causing mutations. Each individual (singleton) exome harbors over 500 rare variants of unknown significance (VUS) in the splicing region. The existing relevant pathogenicity prediction tools tackle all non-coding variants as one amorphic class and/or are not calibrated for the high sensitivity required for clinical use. Here we calibrate seven such tools and devise a novel tool called Splicing Clinically Applicable Pathogenicity prediction (S-CAP) that is over twice as powerful as all previous tools, removing 41% of patient VUS at 95% sensitivity. We show that S-CAP does this by using its own features and not via meta-prediction over previous tools, and that splicing pathogenicity prediction is distinct from predicting molecular splicing changes. S-CAP is an important step on the path to deriving non-coding causal diagnoses.


Assuntos
Variação Genética/genética , Processamento de RNA/genética , Exoma/genética , Humanos , Mutação/genética
20.
Nucleic Acids Res ; 47(5): e27, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30624635

RESUMO

Transposable elements (TEs) are interspersed repeat sequences that make up much of the human genome. Their expression has been implicated in development and disease. However, TE-derived RNA-seq reads are difficult to quantify. Past approaches have excluded these reads or aggregated RNA expression to subfamilies shared by similar TE copies, sacrificing quantitative accuracy or the genomic context necessary to understand the basis of TE transcription. As a result, the effects of TEs on gene expression and associated phenotypes are not well understood. Here, we present Software for Quantifying Interspersed Repeat Expression (SQuIRE), the first RNA-seq analysis pipeline that provides a quantitative and locus-specific picture of TE expression (https://github.com/wyang17/SQuIRE). SQuIRE is an accurate and user-friendly tool that can be used for a variety of species. We applied SQuIRE to RNA-seq from normal mouse tissues and a Drosophila model of amyotrophic lateral sclerosis. In both model organisms, we recapitulated previously reported TE subfamily expression levels and revealed locus-specific TE expression. We also identified differences in TE transcription patterns relating to transcript type, gene expression and RNA splicing that would be lost with other approaches using subfamily-level analyses. Altogether, our findings illustrate the importance of studying TE transcription with locus-level resolution.


Assuntos
Elementos de DNA Transponíveis/genética , Loci Gênicos/genética , Análise de Sequência de RNA/métodos , Software , Transcrição Genética/genética , Esclerose Amiotrófica Lateral/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Camundongos , Processamento de RNA/genética
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