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1.
J Nutr Sci Vitaminol (Tokyo) ; 68(4): 284-293, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36047100

RESUMO

Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.


Assuntos
Fosfatase Alcalina , Proteínas Ligadas por GPI , Intestinos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Processamento Alternativo , Células CACO-2 , Células Epiteliais/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Oxid Med Cell Longev ; 2022: 8645830, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36062189

RESUMO

Background: Covalently closed circular RNAs (circRNAs) play critical oncogenic or anticancer roles in various cancers including renal cell carcinoma (RCC), pointing to their regulation as a promising strategy against development of RCC. We, thus, studied the tumor-suppressive role of circ_000829 in RCC through in vitro and in vivo experiments. Methods: The expression of circ_000829 was validated in clinical RCC tissues and RCC cell lines. Based on ectopic expression and knockdown experiments, we examined the interactions among circ_000829, serine and arginine rich splicing factor 1 (SRSF1), and solute carrier family 39 member 14 (SLC39A14, zinc transporter). Then, the effects of circ_000829, SRSF1, and SLC39A14 on cell cycle distribution and proliferation in vitro and on tumor growth in vivo were evaluated in RCC cells. Results: Circ_000829 was poorly expressed in RCC tissues and cells, while SRSF1 was highly expressed. Restoration of circ_000829 reduced the levels of SRSF1 and SLC39A14B, thereby repressing the RCC cell proliferation in vitro and tumor growth in vivo. Meanwhile, overexpression of SRSF1 and SLC39A14B promoted the proliferation and cell cycle entry of RCC cells. Mechanistically, circ_000829 directly bound to SRSF1, and SRSF1 enhanced the expression of SLC39A14B by mediating the alternative splicing of SLC39A14. SLC39A14B upregulation negated the effect of SLC39A14 knockdown on RCC cell proliferation. Conclusion: Hence, this study suggests the antiproliferative role of circ_000829 in RCC growth and further elucidates the underlying mechanism involving the inhibited SRSF1-mediated alternative splicing of SLC39A14 mRNA.


Assuntos
Carcinoma de Células Renais , Proteínas de Transporte de Cátions , Neoplasias Renais , RNA Circular , Fatores de Processamento de Serina-Arginina , Processamento Alternativo , Carcinoma de Células Renais/patologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/patologia , RNA Circular/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
3.
BMC Plant Biol ; 22(1): 432, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36076169

RESUMO

BACKGROUND: Salicylic acid (SA) is a phytohormone which works to regulate the abiotic stress response of plants. However, the molecular mechanism by which SA mediates heat tolerance in waxy maize (Zea mays L. sinsensis Kulesh) remains unknown. RESULTS: Two varieties of waxy maize seedlings, heat-tolerant 'Yunuo7' (Y7) and heat-sensitive 'Suyunuo5' (S5), were pretreated with SA prior to heat stress (HTS). After treatment, physiological and transcriptomic changes were analyzed. Compared with HTS, the exogenous application of SA enhanced the shoot dry weight, the activities of antioxidant enzymes (e.g., SOD, POD, CAT and APX), and the concentration of endogenous phytohormones (e.g., SA, ABA, IAA, GA3), while decreased the MDA content. Transcriptome analysis showed that the number of differentially expressed genes (DEGs) identified in the control (CK) vs HTS and HTS vs HTS + SA comparisons were more in S5 than in Y7. HTS induced the downregulation of genes involved in photosynthesis and the upregulation of genes encoding heat shock transcription factors (HSFs) and heat shock proteins (HSPs). Compared with HTS, SA pretreatment reversed the expression of 5 photosynthesis-related genes, 26 phytohormone-related genes, and all genes encoding HSFs and HSPs in S5. Furthermore, the number of alternative splicing (AS) events increased under HTS treatment for both varieties, while decreased under SA pretreatment of S5. Differentially spliced genes (DSGs) showed little overlap with DEGs, and DEGs and DSGs differed significantly in functional enrichment. CONCLUSIONS: Physiological and transcriptional together indicated that HTS and SA pretreatment had a greater effect on S5 than Y7. Additionally, it appears that transcriptional regulation and AS work synergistically to enhance thermotolerance in heat-sensitive waxy maize. Our study revealed the regulatory effects and underlying molecular mechanisms of SA on waxy maize seedling under HTS.


Assuntos
Plântula , Zea mays , Processamento Alternativo , Resposta ao Choque Térmico/genética , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Plântula/fisiologia , Transcriptoma , Ceras/metabolismo , Zea mays/metabolismo
4.
Molecules ; 27(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36080449

RESUMO

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.


Assuntos
Processamento Alternativo , Oligonucleotídeos Antissenso , Processamento Alternativo/genética , Éxons , Ribonucleoproteína Nuclear Heterogênea A1/genética , Oligonucleotídeos Antissenso/genética , Splicing de RNA
5.
Planta ; 256(4): 72, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36083517

RESUMO

MAIN CONCLUSION: SR proteins from sweet potato have conserved functional domains and similar gene structures as that of Arabidopsis and rice in general. However, expression patterns and alternative splicing regulations of SR genes from different species have changed under stresses. Novel alternative splicing regulations were found in sweet potato SR genes. Serine/arginine-rich (SR) proteins play important roles in plant development and stress response by regulating the pre-mRNA splicing process. However, SR proteins have not been identified so far from an important crop sweet potato. Through bioinformatics analysis, our study identified 24 SR proteins from sweet potato, with comprehensively analyzing of protein characteristics, gene structure, chromosome localization, and cis-acting elements in promotors. Salt, heat, and mimic drought stresses triggered extensive but different expressional regulations on sweet potato SR genes. Interestingly, heat stress caused the most active disturbances in both gene transcription and pre-mRNA alternative splicing (AS). Tissue and species-specific transcriptional and pre-mRNA AS regulations in response to stresses were found in sweet potato, in comparison with Arabidopsis and rice. Moreover, novel patterns of pre-mRNA alternative splicing were found in SR proteins from sweet potato. Our study provided an insight into similarities and differences of SR proteins in different plant species from gene sequences to gene structures and stress responses, indicating SR proteins may regulate their downstream genes differently between different species and tissues by varied transcriptional and pre-mRNA AS regulations.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ipomoea batatas , Oryza , Processamento Alternativo/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ipomoea batatas/genética , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Precursores de RNA/genética
6.
Biochem Biophys Res Commun ; 628: 25-31, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36063599

RESUMO

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5' and 3' region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of ß-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.


Assuntos
Quimotripsina , Inibidores de Serino Proteinase , Processamento Alternativo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Catepsina G/metabolismo , Quimases/metabolismo , Quimotripsina/metabolismo , Códon de Terminação , Citocinas/metabolismo , Humanos , Nucleotídeos/metabolismo , Isoformas de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Inibidores de Serino Proteinase/química
7.
BMC Res Notes ; 15(1): 286, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064446

RESUMO

OBJECTIVE: Osteopontin (OPN) is a well-known glycoprotein involved in numerous pathobiological processes, including cancer. Despite having five splice variants for osteopontin in mice, the main focus of most studies has been on total OPN (tOPN). There are some studies on other splice variants, but the expression of osteopontin-5 (OPN5) has not been addressed in mouse cancer cells. Therefore, this study sought to evaluate OPN5 expression in mouse breast cancer cells. RESULTS: The expression of OPN5 in primary and metastatic breast cancer cells of mice was confirmed in our study. These findings provided important insights regarding the OPN alternative splicing in mice for the first time. It is concluded that, like other OPN-SVs, OPN5 probably plays an essential role in tumor progression, which requires further investigation in different tumor models.


Assuntos
Neoplasias , Osteopontina , Processamento Alternativo , Animais , Proteínas de Membrana/metabolismo , Camundongos , Opsinas/metabolismo , Osteopontina/genética , Osteopontina/metabolismo
8.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077545

RESUMO

Serine/arginine-rich (SR) proteins are a type of splicing factor. They play significant roles in constitutive and alternative pre-mRNA splicing, and are involved in post-splicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, mRNA translation, and miRNA biogenesis. In plants, SR proteins function under a complex regulatory network by protein-protein and RNA-protein interactions between SR proteins, other splicing factors, other proteins, or even RNAs. The regulatory networks of SR proteins are complex-they are regulated by the SR proteins themselves, they are phosphorylated and dephosphorylated through interactions with kinase, and they participate in signal transduction pathways, whereby signaling cascades can link the splicing machinery to the exterior environment. In a complex network, SR proteins are involved in plant growth and development, signal transduction, responses to abiotic and biotic stresses, and metabolism. Here, I review the current status of research on plant SR proteins, construct a model of SR proteins function, and ask many questions about SR proteins in plants.


Assuntos
Arginina , Serina , Processamento Alternativo , Arginina/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Precursores de RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
9.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077555

RESUMO

Pairing of splice sites across an intron or exon is the central point of intron or exon definition in pre-mRNA splicing with the latter mode proposed for most mammalian exons. However, transcriptome-wide pairing within endogenous transcripts has not been examined for the prevalence of each mode in mammalian cells. Here we report such pairings in rat GH3 pituitary cells by measuring the relative abundance of nuclear RNA-Seq reads at the intron start or end (RISE). Interestingly, RISE indexes are positively correlated between 5' and 3' splice sites specifically across introns or exons but inversely correlated with the usage of adjacent exons. Moreover, the ratios between the paired indexes were globally modulated by depolarization, which was disruptible by 5-aza-Cytidine. The nucleotide matrices of the RISE-positive splice sites deviate significantly from the rat consensus, and short introns or exons are enriched with the cross-intron or -exon RISE pairs, respectively. Functionally, the RISE-positive genes cluster for basic cellular processes including RNA binding/splicing, or more specifically, hormone production if regulated by depolarization. Together, the RISE analysis identified the transcriptome-wide regulation of either intron or exon definition between weak splice sites of short introns/exons in mammalian cells. The analysis also provides a way to further track the splicing intermediates and intron/exon definition during the dynamic regulation of alternative splicing by extracellular factors.


Assuntos
Precursores de RNA , Transcriptoma , Processamento Alternativo , Animais , Éxons/genética , Íntrons/genética , Mamíferos/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Ratos
10.
Sci Adv ; 8(33): eabn9232, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35977015

RESUMO

Dysregulation of alternative splicing is a key molecular hallmark of cancer. However, the common features and underlying mechanisms remain unclear. Here, we report an intriguing length-dependent splicing regulation in cancers. By systematically analyzing the transcriptome of thousands of cancer patients, we found that short exons are more likely to be mis-spliced and preferentially excluded in cancers. Compared to other exons, cancer-associated short exons (CASEs) are more conserved and likely to encode in-frame low-complexity peptides, with functional enrichment in GTPase regulators and cell adhesion. We developed a CASE-based panel as reliable cancer stratification markers and strong predictors for survival, which is clinically useful because the detection of short exon splicing is practical. Mechanistically, mis-splicing of CASEs is regulated by elevated transcription and alteration of certain RNA binding proteins in cancers. Our findings uncover a common feature of cancer-specific splicing dysregulation with important clinical implications in cancer diagnosis and therapies.


Assuntos
Processamento Alternativo , Neoplasias , Éxons , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Fases de Leitura , Transcriptoma
11.
Retrovirology ; 19(1): 18, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35986377

RESUMO

BACKGROUND: The generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs). METHODS: Both shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression. Effects on virus expression were assessed using western blotting, RT-qPCR, and immunofluorescence. RESULTS: The studies demonstrate that SR kinases play distinct roles; depletion of CLK1 enhanced HIV-1 gene expression, reduction of CLK2 or SRPK1 suppressed it, whereas CLK3 depletion had a modest impact. The opposing effects of CLK1 vs. CLK2 depletion were due to action at distinct steps; reduction of CLK1 increased HIV-1 promoter activity while depletion of CLK2 affected steps after transcript initiation. Reduced CLK1 expression also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine whether small molecule modulation of SR kinase function could be used to control HIV-1 replication, we screened a GSK library of protein kinase inhibitors (PKIS) and identified several pyrazolo[1,5-b] pyridazine derivatives that suppress HIV-1 gene expression/replication with an EC50 ~ 50 nM. The compounds suppressed HIV-1 protein and viral RNA accumulation with minimal impact on cell viability, inhibiting CLK1 and CLK2 but not CLK3 function, thereby selectively altering the abundance of individual CLK and SR proteins in cells. CONCLUSIONS: These findings demonstrate the unique roles played by individual SR kinases in regulating HIV-1 gene expression, validating the targeting of these functions to either enhance latency reversal, essential for "Kick-and-Kill" strategies, or to silence HIV protein expression for "Block-and-Lock" strategies.


Identifying cellular factors that regulate HIV-1 RNA processing provides important insights into novel strategies to control this infection. Different members of the SR kinase family have distinct roles in regulating virus expression because they affect distinct steps of transcription/RNA processing. We identify inhibitors of these kinases that suppress HIV-1 gene expression and replication in multiple assay systems at nanomolar concentrations with limited or no cytotoxicity. Our results highlight the therapeutic potential of targeting the post-integration stage of the HIV-1 lifecycle to selectively enhance or reverse provirus latency. A greater understanding of the molecular mechanisms underlying the effects observed will facilitate the development of more targeted approaches to modulate HIV-1 latency on the path toward a "functional" cure for this infection.


Assuntos
HIV-1 , Processamento Alternativo , Expressão Gênica , HIV-1/fisiologia , Inibidores de Proteínas Quinases/farmacologia , RNA Viral/genética , Latência Viral
12.
Mol Genet Genomics ; 297(5): 1439-1449, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35939099

RESUMO

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.


Assuntos
Sítios de Splice de RNA , Retinite Pigmentosa , Processamento Alternativo , Códon de Terminação , Proteínas do Citoesqueleto , Humanos , Mutação , Mutação de Sentido Incorreto , Splicing de RNA , RNA Mensageiro
13.
Nature ; 608(7922): 353-359, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35922509

RESUMO

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.


Assuntos
Alelos , Perfilação da Expressão Gênica , Especificidade de Órgãos , RNA-Seq , Transcriptoma , Processamento Alternativo/genética , Linhagem Celular , Conjuntos de Dados como Assunto , Genótipo , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Especificidade de Órgãos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/deficiência , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Reprodutibilidade dos Testes , Transcriptoma/genética
14.
Front Immunol ; 13: 939863, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35979358

RESUMO

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Splicing de RNA , Proteínas dos Retroviridae/metabolismo , Adulto , Processamento Alternativo , RNA Helicases DEAD-box/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Proteínas Virais/metabolismo
15.
G3 (Bethesda) ; 12(9)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35920767

RESUMO

Genetic disruptions to the biogenesis of spliceosomal small-nuclear ribonucleoproteins in Drosophila cause wide-spread alternative splicing changes, including changes to the splicing of pre-mRNA for Ribosomal protein S21 (RpS21). Using a transposon mutant for the Phosphorylated adaptor for RNA export (Phax) gene, we demonstrate that changes in the splicing of RpS21 transcripts have a strong influence on the developmental progression of PhaxSH/SH mutants. Different alleles of the Drosophila RpS21 gene are circulating in common laboratory strains and cell lines. These alleles exhibit differences in RpS21 intron retention and splicing efficiency. Differences in the splicing of RpS21 transcripts account for prior conflicting observations of the phenotypic severity of PhaxSH/SH mutant stocks. The alleles uncover a strong splicing enhancer in RpS21 transcripts that can fully suppress the larval lethality and partially suppress the pupal lethality exhibited by PhaxSH/SH mutant lines. In the absence of the splicing enhancer, the splicing of RpS21 transcripts can be modulated in trans by the SR-rich B52 splicing factor. As PhaxSH/SH mutants exhibit wide-spread splicing changes in transcripts for other genes, findings here establish the importance of a single alternative splicing event, RpS21 splicing or intron retention, to the developmental progression of Drosophila.


Assuntos
Proteínas de Drosophila , Drosophila , Alelos , Processamento Alternativo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutação , RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Ribossômicas
17.
PLoS Genet ; 18(8): e1010342, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35926060

RESUMO

Genes generate transcripts of various functions by alternative splicing. However, in most transcriptome studies, short-reads sequencing technologies (next-generation sequencers) have been used, leaving full-length transcripts unobserved directly. Although long-reads sequencing technologies would enable the sequencing of full-length transcripts, the data analysis is difficult. In this study, we developed an analysis pipeline named SPLICE and analyzed cDNA sequences from 42 pairs of hepatocellular carcinoma (HCC) and matched non-cancerous livers with an Oxford Nanopore sequencer. Our analysis detected 46,663 transcripts from the protein-coding genes in the HCCs and the matched non-cancerous livers, of which 5,366 (11.5%) were novel. A comparison of expression levels identified 9,933 differentially expressed transcripts (DETs) in 4,744 genes. Interestingly, 746 genes with DETs, including the LINE1-MET transcript, were not found by a gene-level analysis. We also found that fusion transcripts of transposable elements and hepatitis B virus (HBV) were overexpressed in HCCs. In vitro experiments on DETs showed that LINE1-MET and HBV-human transposable elements promoted cell growth. Furthermore, fusion gene detection showed novel recurrent fusion events that were not detected in the short-reads. These results suggest the efficiency of full-length transcriptome studies and the importance of splicing variants in carcinogenesis.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Processamento Alternativo/genética , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Elementos de DNA Transponíveis , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Hepáticas/genética , Splicing de RNA/genética , Transcriptoma/genética
18.
Nat Commun ; 13(1): 4659, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36002455

RESUMO

Splicing quantitative trait loci (sQTLs) are one of the major causal mechanisms in genome-wide association study (GWAS) loci, but their role in disease pathogenesis is poorly understood. One reason is the complexity of alternative splicing events producing many unknown isoforms. Here, we propose two approaches, namely integration and selection, for this complexity by focusing on protein-structure of isoforms. First, we integrate isoforms with the same coding sequence (CDS) and identify 369-601 integrated-isoform ratio QTLs (i2-rQTLs), which altered protein-structure, in six immune subsets. Second, we select CDS incomplete isoforms annotated in GENCODE and identify 175-337 isoform-ratio QTL (i-rQTL). By comprehensive long-read capture RNA-sequencing among these incomplete isoforms, we reveal 29 full-length isoforms with unannotated CDSs associated with GWAS traits. Furthermore, we show that disease-causal sQTL genes can be identified by evaluating their trans-eQTL effects. Our approaches highlight the understudied role of protein-altering sQTLs and are broadly applicable to other tissues and diseases.


Assuntos
Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Processamento Alternativo/genética , Suscetibilidade a Doenças , Humanos , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Locos de Características Quantitativas/genética
19.
Mol Cell ; 82(16): 2982-2999.e14, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35914530

RESUMO

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.


Assuntos
Neurogênese , Splicing de RNA , Processamento Alternativo , Animais , Éxons/genética , Mamíferos , Camundongos , Neurogênese/genética , Neurônios , Proteínas de Ligação a RNA/genética
20.
RNA Biol ; 19(1): 1007-1018, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35980273

RESUMO

Ovarian cancer (OV) is characterized by high incidence and poor prognosis. Increasing evidence indicates that aberrant alternative splicing (AS) events are associated with the pathogenesis of cancer. We examined prognosis-related alternative splicing events and constructed a clinically applicable model to predict patients' outcomes. Public database including The Cancer Genome Atlas (TCGA), TCGA SpliceSeq, and the Genomics of Drug Sensitivity in Cancer databases were used to detect the AS expression, immune cell infiltration and IC50. The prognosis-related AS model was constructed and validated by using Cox regression, LASSO regression, C-index, calibration plots, and ROC curves. A total of eight AS events (including FLT3LG|50942|AP) were selected to establish the prognosis-related AS model. Compared with high-risk group, low-risk group had a better outcome (P = 1.794e-06), was more sensitive to paclitaxel (P = 0.022), and higher proportions of plasma cells. We explored the upstream regulatory mechanisms of prognosis-related AS and found that two splicing factor and 156 tag single nucleotide polymorphisms may be involved in the regulation of prognosis-related AS. In order to assess patient prognosis more comprehensively, we constructed a clinically applicable model combining risk score and clinicopathological features, and the 1 -, and 3-year AUCs of the clinically applicable model were 0.812, and 0.726, which were 7.5% and 3.3% higher than that of the risk score. We constructed a prognostic signature for OV patients and comprehensively analysed the regulatory characteristics of the prognostic AS events in OV.


Assuntos
Processamento Alternativo , Neoplasias Ovarianas , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Ovarianas/genética
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