RESUMO
All living organisms must respond to, and defend against, environmental stresses. Depending on the extent and severity of stress, cells try to alter their metabolism and adapt to a new state. Changes in alternative splicing of pre-mRNA are a crucial regulation mechanism through which cells are able to respond to a decrease in oxygen tension in the cellular environment. Currently, only limited data are available in the literature on how short-term hypoxia influences mRNA isoform formation. In this work, we discovered that expressions of the same genes that are activated during cellular stress are also activated in cells under short-term hypoxic conditions. Our results demonstrate that short-term hypoxia influences the splicing of genes associated with cell stress and apoptosis; however, the mRNA isoform formation patterns from the same pre-mRNAs in cells under short-term hypoxic conditions and prolonged hypoxia are different. Obtained data also show that short-term cellular hypoxia increases protein phosphatase but not protein kinase expression. Enhanced levels of protein phosphatase expression in cells are clearly important for changing mRNA isoform formation.
Assuntos
Isoformas de RNA , Precursores de RNA , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo , Fosfoproteínas Fosfatases , Isoformas de Proteínas/genética , Precursores de RNA/genéticaRESUMO
Alternative pre-mRNA splicing plays a very important role in expanding protein diversity as it generates numerous transcripts from a single protein-coding gene. Therefore, alterations lead this process to neurological human disorders, including Alzheimer's and Parkinson's diseases. Moreover, accumulating evidence indicates that the splicing machinery highly contributes to the cells' ability to adapt to different altered cellular microenvironments, such as hypoxia. Hypoxia is known to have an effect on the expression of proteins involved in a multiple of biological processes, such as erythropoiesis, angiogenesis, and neurogenesis, and is one of the important risk factors in neuropathogenesis. In this review, we discuss the current knowledge of alternatively spliced genes, which, as it is reported, are associated with Alzheimer's and Parkinson's diseases. Additionally, we highlight the possible influence of cellular hypoxic microenvironment for the formation of mRNA isoforms contributing to the development of these neurodegenerative diseases.
Assuntos
Processamento Alternativo/genética , Doença de Alzheimer/genética , Hipóxia/genética , Doença de Parkinson/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Neurogênese/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Splicing de RNA/genéticaRESUMO
The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia are involved in large-scale alterations in alternative pre-mRNA splicing. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. In the present study we show that U2AF is involved in hypoxia dependent anti-apoptotic Fas mRNA isoform formation. Our performed studies show that U2AF-RNA interaction is reduced in hypoxic cells, leading to reduction of Fas and increased sFas mRNAs formation. Efficient U2AF-RNA interactions of both subunits are important for Fas exon 6 inclusion into forming mRNA in normoxic and hypoxic cells.
Assuntos
Hipóxia/genética , Fator de Processamento U2AF/fisiologia , Receptor fas/genética , Processamento Alternativo/genética , Células HCT116 , Humanos , Hipóxia/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Processamento U2AF/genética , Receptor fas/metabolismoRESUMO
The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer's disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer's disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Hipóxia Celular/genética , RNA Mensageiro/genética , Proteínas tau/genética , Processamento Alternativo/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Íntrons/genética , Precursores de RNA/genética , Transcrição Gênica/genéticaRESUMO
The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia involve large-scale alterations in alternative pre-mRNA splicing. Cancer associated Fas protein plays a central role in the physiological regulation of programmed cell death and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. Another cancer related protein Rac1 plays an important regulatory role specifically in cells' motility, growth and survival. Rac pre-mRNA is alternatively spliced to produce Rac1b protein, which is upregulated in metastatic diseases. In the present study we, for the first time, show that anti-apoptotic Fas mRNA isoform formation is regulated by cellular microenvironment - hypoxia. Hypoxic microenvironment, however, does not influence Rac1 pre-mRNAs alternative splicing. Also our presented results indicate that splicing factors hnRNP A1 and SPF45, previously shown to regulate Fas alternative splicing in normoxic cells, are not involved in hypoxia dependent alternative Fas pre-mRNA splicing regulation in an amount dependent manner. Our observations on hypoxia dependent alternative Fas pre-mRNA splicing regulation indicate a probable involvement of other, yet unidentified splicing factors. Presented data also shows the contribution of pre-mRNA splicing to cell survival under unfavorable conditions.
Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/genética , Neoplasias/genética , Fatores de Processamento de RNA/genética , Receptor fas/genética , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Íntrons/genética , Neoplasias/patologia , Precursores de RNA/genética , Splicing de RNA/genética , Hipóxia Tumoral/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: An abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered. METHODS: We experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5'- and 3'-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells. RESULTS: We found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells. CONCLUSIONS: An interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer.
Assuntos
Neoplasias do Sistema Digestório/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Renais/genética , MicroRNAs/genética , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Linhagem Celular Tumoral , Neoplasias do Sistema Digestório/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Neoplasias Renais/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
BACKGROUND: Cell lines derived from human tumors have been extensively used as experimental models of neoplastic disease. Although such cell lines differ from both normal and cancerous tissue. OBJECTIVE: The data obtained used DNA and RNA microarray systems does not give full information about protein expression levels in cells and tissues. We present experimental evidence that splicing factor SRSF1, SRSF2, U2AF35, U2AF65 and KHSRP expression levels in gastrointestinal tract (colon, gastric and pancreatic) tumors differ compare to healthy tissues and in cell lines, derived from corresponding organs. METHODS: Protein expression was analyzed using Western blots. RT-PCR method was used for Fas and Rac splicing analysis. RESULTS: Obtained results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumors in vivo. Expression levels of individual splicing factors in tumors might serve as tumor markers. Not all experimental results obtained from cell lines reflect changes that occur in tumors. Also Fas and Rac, cancer associated genes, tumor associated sFas and Rac1b mRNA isoform profiles in cell lines do not correspond to profiles that are observed in tumors. CONCLUSIONS: Not all experimental results obtained in cell lines reflect changes that occur in real tumors.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Neoplasias Gastrointestinais/genética , Isoformas de RNA/genética , Fatores de Transcrição/biossíntese , Transcrição Gênica , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Isoformas de RNA/biossíntese , Fatores de Processamento de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles.
Assuntos
Processamento Alternativo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Arginina/genética , Arginina/metabolismo , Hipóxia Celular , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Precursores de RNA/metabolismo , Serina/genética , Serina/metabolismoRESUMO
Monoamine oxidase B (MAO-B) plays an important role in the metabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues. Increased levels of MAO-B mRNA and enzymatic activity have been reported in platelets from patients with Parkinson's and Alzheimer's diseases, however the triggers of enhanced mRNA levels are unknown. Our results demonstrate for the first time that G/A dimorphism in intron 13 sequence creates splicing enhancer thus stimulating intron 13 removal efficiency. The increased MAO-B protein levels might serve as a surrogate marker for - Parkinson disease.