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1.
Wiley Interdiscip Rev RNA ; : e1828, 2023 Nov 23.
Article in English | MEDLINE | ID: mdl-37994271

ABSTRACT

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in numerous biological processes. The arachidonic acid (AA) metabolic pathway is a fundamental biochemical pathway responsible for the enzymatic conversion of AA, a 20-carbon omega-six polyunsaturated fatty acid, into a variety of potent lipid signaling molecules known as eicosanoids. Eicosanoids are produced through the cyclooxygenase and lipoxygenase arms of the AA pathway and have diverse biological roles in both healthy and disease states, including cancer and inflammatory diseases. Cyclooxygenase 2 (COX-2), the inducible, rate-limiting enzyme of the cyclooxygenase arm, produces two main forms of eicosanoids: prostaglandins and thromboxanes. AA  metabolized through the lipoxygenase arm by the action of 5-lipoxygenase (ALOX5) produces eicosanoids known as leukotrienes. COX-2 and ALOX5 gene expression are regulated through many different lncRNAs and microRNA (miRNA)-mediated mechanisms. As previously reviewed, noncoding RNAs affect transcription, splicing, alternative polyadenylation, messenger RNA stability, translation, and miRNA regulation of COX-2 and ALOX5 (Lutz and Cornett, 2013, Wiley Interdisciplinary Reviews. RNA, 4(5), 593-605). This current review discusses the intricate roles of lncRNAs, including MALAT1, NEAT1, HOTAIR, PACER, and others, in modulating the AA pathway. In this review update, we will delve into advancements in our understanding of AA gene expression regulation. We will explore the mechanisms of lncRNAs and their associated miRNAs and proteins known to regulate key components of the AA signaling pathway. We will also discuss the therapeutic potential of targeting lncRNA-mediated regulation, with a focus on modulating COX-2 and ALOX5 activity and downstream eicosanoid production for applications in inflammatory and oncological conditions. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease.

2.
Oncotarget ; 13: 291-306, 2022.
Article in English | MEDLINE | ID: mdl-35136486

ABSTRACT

Long noncoding RNAs (lncRNAs) are known to regulate gene expression; however, in many cases, the mechanism of this regulation is unknown. One novel lncRNA relevant to inflammation and arachidonic acid (AA) metabolism is the p50-associated COX-2 extragenic RNA (PACER). We focused our research on the regulation of PACER in lung cancer. While the function of PACER is not entirely understood, PACER is known to play a role in inflammation-associated conditions. Our data suggest that PACER is critically involved in COX-2 transcription and dysregulation in lung cancer cells. Our analysis of The Cancer Genome Atlas (TCGA) expression data revealed that PACER expression is significantly higher in lung adenocarcinomas than normal lung tissues. Additionally, we discovered that elevated PACER expression strongly correlates with COX-2 expression in lung adenocarcinoma patients. Specific siRNA-mediated knockdown of PACER decreases COX-2 expression indicating a direct relationship. Additionally, we show that PACER expression is induced upon treatment with proinflammatory cytokines to mimic inflammation. Treatment with prostaglandin E2 (PGE2) induces both PACER and COX-2 expression, suggesting a PGE2-mediated feedback loop. Inhibition of COX-2 with celecoxib decreased PACER expression, confirming this self-regulatory process. Significant overlap between the COX-2 promotor and the PACER promotor led us to investigate their transcriptional regulatory mechanisms. Treatment with pharmacologic inhibitors of NF-κB or AP-1 showed a modest effect on both PACER and COX-2 expression but did not eliminate expression. These data suggest that the regulation of expression of both PACER and COX-2 is complex and intricately linked.


Subject(s)
Lung Neoplasms , RNA, Long Noncoding , Arachidonic Acid/metabolism , Celecoxib , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Dinoprostone/metabolism , Humans , Inflammation/metabolism , Lung/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , NF-kappa B/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Small Interfering/metabolism , Transcription Factor AP-1/metabolism
3.
Mediators Inflamm ; 2021: 5595520, 2021.
Article in English | MEDLINE | ID: mdl-33776573

ABSTRACT

Two pathways commonly dysregulated in autoimmune diseases and cancer are tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1ß) signaling. Researchers have also shown that both signaling cascades positively regulate arachidonic acid (AA) signaling. More specifically, TNFα/IL-1ß promotes expression of the prostaglandin E2- (PGE2-) producing enzymes, cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1). Exacerbated TNFα, IL-1ß, and AA signaling have been associated with many diseases. While some TNFα therapies have significantly improved patients' lives, there is still an urgent need to develop novel therapeutics that more comprehensively treat inflammatory-related diseases. Recently, researchers have begun to use RNA interference (RNAi) to treat various diseases in the clinic. One type of RNAi is microRNA (miRNA), a class of small noncoding RNA found within cells. One miRNA in particular, miR-708, has been shown to target COX-2 and mPGES-1. Previous studies have also suggested that miR-708 may be a negative regulator of TNFα/IL-1ß signaling. Therefore, we studied the relationship between miR-708, TNFα/IL-1ß, and AA signaling in diseased lung cells. We found that miR-708 negatively regulates TNFα/IL-1ß signaling in nondiseased lung cells, which is lost in diseased lung cells. Transient transfection of miR-708 suppressed TNFα/IL-1ß-induced changes in COX-2, mPGES-1, and PGE2 levels. Moreover, miR-708 also suppressed TNFα/IL-1ß-induced IL-6 independent of AA signaling. Mechanistically, we determined that miR-708 suppressed IL-6 signaling by reducing expression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activator inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß). Collectively, our data suggest miR-708 regulates TNFα/IL-1ß signaling by inhibiting multiple points of the signaling cascade.


Subject(s)
Arachidonic Acid/metabolism , Interleukin-1beta/metabolism , MicroRNAs/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , A549 Cells , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , MicroRNAs/genetics , Nitriles/pharmacology , Signal Transduction/drug effects , Sulfones/pharmacology
4.
Oncotarget ; 11(26): 2464-2483, 2020 Jun 30.
Article in English | MEDLINE | ID: mdl-32655834

ABSTRACT

Many cancers maintain an inflammatory microenvironment to promote their growth. Lung cancer is of particular importance, as it is the deadliest cancer worldwide. One inflammatory pathway commonly dysregulated in cancer is the metabolism of arachidonic acid (AA) by Cyclooxygenase-2 (COX-2) and microsomal Prostaglandin E Synthase 1 (mPGES-1) into Prostaglandin E2 (PGE2). While researchers have identified PGE2's pro-tumorigenic functions, the mechanisms governing overexpression of COX-2 and mPGES-1 are incompletely understood. MicroRNAs (miRNAs) are important post-transcriptional regulators commonly dysregulated in cancer. Interestingly, miR-708-5p (miR-708) is predicted to target both COX-2 and mPGES-1. In this study, we show that high miR-708 expression is associated with survival rates in lung squamous cell carcinoma patients. miR-708 also represses PGE2 production by suppressing both COX-2 and mPGES-1 expression in lung cancer cells. miR-708 regulation of COX-2 and mPGES-1 is mediated through targeting of their 3' untranslated regions (UTRs). Moreover, miR-708 decreases proliferation, survival, and migration of lung cancer cells, which can be partially attributed to miR-708's inhibition of PGE2 signaling. Lastly, we identify novel miR-708 predicted targets and possible regulators of miR-708 expression in lung cancer. Collectively, these data demonstrate that dysregulated miR-708 expression contributes to exacerbated PGE2 production, leading to an enhanced pro-tumorigenic phenotype in lung cancer cells.

5.
Oncotarget ; 11(51): 4699-4721, 2020 Dec 22.
Article in English | MEDLINE | ID: mdl-33473256

ABSTRACT

Lung cancer is a collection of aggressive tumors generally not diagnosed until late-stage, resulting in high mortality rates. The vast majority of non-small cell lung cancer (NSCLC) patients undergo combinatory chemotherapeutic treatment, which initially reduces tumor growth, but frequently becomes ineffective due to toxicity and resistance. Researchers have identified multiple signaling pathways involved in lung cancer chemoresistance, including cyclooxygenase-2 (COX-2)/microsomal prostaglandin E synthase-1 (mPGES-1) derived prostaglandin E2 (PGE2). While COX-2 inhibitors have shown promise in the clinic, their use is limited due to severe side effects. One novel approach to effectively suppress COX-2 signaling is through microRNA (miRNA). MiRNAs are small-noncoding RNAs commonly misexpressed in cancer. One tumor suppressive miRNA, miR-708-5p, has been shown to repress pro-resistant signaling pathways, including COX-2 and mPGES-1. Here, we demonstrate that chemotherapies reduce COX-2 expression, possibly through induction of miR-708-5p. Moreover, combination treatment of erlotinib (ERL) or paclitaxel (PAC) with miR-708-5p enhances COX-2 and mPGES-1 protein suppression. We also show that combination chemotherapeutic and miR-708-5p treatment intensifies the anti-proliferative and pro-apoptotic effects of ERL and PAC. We also created ERL and PAC resistant lung cancer cell lines, which have increased COX-2 expression and diminished miR-708-5p levels compared to naïve lung cancer cells. While ERL and PAC treatments do not alter resistant cell phenotype alone, combination treatment with miR-708-5p partially restores the chemotherapies' anti-proliferative effects and fully restores their pro-apoptotic qualities. These data suggest miR-708-5p may have potential combinatory therapeutic value to more efficaciously treat lung tumors while overcoming chemoresistance.

6.
RNA Biol ; 16(12): 1721-1732, 2019 12.
Article in English | MEDLINE | ID: mdl-31425002

ABSTRACT

Non-small cell lung cancer (NSCLC) is a complex disease in need of new methods of therapeutic intervention. Recent interest has focused on using microRNAs (miRNAs) as a novel treatment method for various cancers. miRNAs negatively regulate gene expression post-transcriptionally, and have become attractive candidates for cancer treatment because they often simultaneously target multiple genes of similar biological function. One such miRNA is miR-146a-5p, which has been described as a tumor suppressive miRNA in NSCLC cell lines and tissues. In this study, we performed RNA-Sequencing (RNA-Seq) analysis following transfection of synthetic miR-146a-5p in an NSCLC cell line, A549, and validated our data with Gene Ontology and qRT-PCR analysis of known miR-146a-5p target genes. Our transcriptomic data revealed that miR-146a-5p exerts its tumor suppressive function beyond previously reported targeting of EGFR and NF-κB signaling. miR-146a-5p mimic transfection downregulated arachidonic acid metabolism genes, the RNA-binding protein HuR, and many HuR-stabilized pro-cancer mRNAs, including TGF-ß, HIF-1α, and various cyclins. miR-146a-5p transfection also reduced expression and cellular release of the chemokine CCL2, and this effect was mediated through the 3' untranslated region of its mRNA. Taken together, our work reveals that miR-146a-5p functions as a tumor suppressor in NSCLC by controlling various metabolic and signaling pathways through direct and indirect mechanisms.


Subject(s)
Adenocarcinoma of Lung/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , MicroRNAs/genetics , Transcriptome , A549 Cells , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/mortality , Adenocarcinoma of Lung/pathology , Animals , Arachidonic Acid/antagonists & inhibitors , Arachidonic Acid/metabolism , Atlases as Topic , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Cyclins/genetics , Cyclins/metabolism , ELAV-Like Protein 1/genetics , ELAV-Like Protein 1/metabolism , Gene Expression Profiling , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Lung Neoplasms/pathology , MicroRNAs/metabolism , Signal Transduction , Survival Analysis , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism
7.
Sci Rep ; 9(1): 5405, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30931980

ABSTRACT

Elevated prostaglandin E2 (PGE2) levels are observed in colorectal cancer (CRC) patients, and this increase is associated with poor prognosis. Increased synthesis of PGE2 in CRC has been shown to occur through COX-2-dependent mechanisms; however, loss of the PGE2-catabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH, HPGD), in colonic tumors contributes to increased prostaglandin levels and poor patient survival. While loss of 15-PGDH can occur through transcriptional mechanisms, we demonstrate that 15-PGDH can be additionally regulated by a miRNA-mediated mechanism. We show that 15-PGDH and miR-21 are inversely correlated in CRC patients, with increased miR-21 levels associating with low 15-PGDH expression. 15-PGDH can be directly regulated by miR-21 through distinct sites in its 3' untranslated region (3'UTR), and miR-21 expression in CRC cells attenuates 15-PGDH and promotes increased PGE2 levels. Additionally, epithelial growth factor (EGF) signaling suppresses 15-PGDH expression while simultaneously enhancing miR-21 levels. miR-21 inhibition represses CRC cell proliferation, which is enhanced with EGF receptor (EGFR) inhibition. These findings present a novel regulatory mechanism of 15-PGDH by miR-21, and how dysregulated expression of miR-21 may contribute to loss of 15-PGDH expression and promote CRC progression via increased accumulation of PGE2.


Subject(s)
Colonic Neoplasms/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Hydroxyprostaglandin Dehydrogenases/genetics , MicroRNAs/genetics , 3' Untranslated Regions/genetics , Binding Sites/genetics , Caco-2 Cells , Cell Proliferation/genetics , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , HCT116 Cells , HT29 Cells , HeLa Cells , Humans , Hydroxyprostaglandin Dehydrogenases/metabolism
8.
Wiley Interdiscip Rev RNA ; 10(4): e1533, 2019 07.
Article in English | MEDLINE | ID: mdl-30895717

ABSTRACT

Cancer as we know it is actually an umbrella term for over 100 very unique malignancies in various tissues throughout the human body. Each type, and even subtype of cancer, has different genetic, epigenetic, and other cellular events responsible for malignant development and metastasis. Recent work has indicated that microRNAs (miRNAs) play a major role in these processes, sometimes by promoting cancer growth and other times by suppressing tumorigenesis. miRNAs are small, noncoding RNAs that negatively regulate expression of specific target genes. This review goes into an in-depth look at the most recent finding regarding the significance of one particular miRNA, miR-146a-5p, and its involvement in cancer. Target gene validation and pathway analysis have provided mechanistic insight into this miRNA's purpose in assorted tissues. Additionally, this review outlines novel findings that suggest miR-146a-5p may be useful as a noninvasive biomarker and as a targeted therapeutic in several cancers. This article is categorized under: RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.


Subject(s)
Carcinogenesis , Cell Transformation, Neoplastic , Gene Expression Regulation , MicroRNAs/metabolism , Neoplasms/physiopathology , Humans
9.
Oncotarget ; 9(42): 26751-26769, 2018 Jun 01.
Article in English | MEDLINE | ID: mdl-29928483

ABSTRACT

Arachidonic acid (AA) can be converted into prostaglandins (PGs) or leukotrienes (LTs) by the enzymatic actions of cyclooxygenases (COX-1 and COX-2) or 5-lipoxygenase (5-LO), respectively. PGs and LTs are lipid signaling molecules that have been implicated in various diseases, including multiple cancers. 5-LO and its activating protein (FLAP) work together in the first two conversion steps of LT production. Previous work has suggested a role for LTs in cancer development and progression. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression post-transcriptionally, and have previously been shown to be involved in cancer. Here, we show that high FLAP expression is associated with lower overall survival in lung adenocarcinoma patients, and FLAP protein is overexpressed in lung cancer cells compared to normal lung cells. Our lab has previously shown that miR-146a regulates COX-2 in lung cancer cells, and this miRNA is also predicted to target FLAP. Transient and stable transfections of miR-146a repress endogenous FLAP expression in lung cancer cells, and reporter assays show this regulation occurs through a direct interaction between the FLAP 3' untranslated region (UTR) and miR-146a. Restoration of miR-146a also results in decreased cancer cell Leukotriene B4 (LTB4) production. Additionally, methylation analysis indicates the miR-146a promoter is hypermethylated in lung cancer cell lines. Taken together, this study and previous work from our lab suggest miR-146a is an endogenous dual inhibitor of AA metabolism in lung cancer cells by regulating both PG and LT production through direct targeting of the COX-2 and FLAP 3' UTRs.

10.
Oncotarget ; 8(41): 71292-71316, 2017 Sep 19.
Article in English | MEDLINE | ID: mdl-29050362

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression post-transcriptionally. They are crucial for normal development and maintaining homeostasis. Researchers have discovered that dysregulated miRNA expression contributes to many pathological conditions, including cancer. miRNAs can augment or suppress tumorigenesis based on their expression and transcribed targetome in various cell types. In recent years, researchers have begun to identify miRNAs commonly dysregulated in cancer. One recently identified miRNA, miR-708-5p, has been shown to have profound roles in promoting or suppressing oncogenesis in a myriad of solid and hematological tumors. This review highlights the diverse, sometimes controversial findings reported for miR-708-5p in cancer, and the importance of further exploring this exciting miRNA.

11.
Inflammation ; 40(2): 645-656, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28124257

ABSTRACT

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cß-2 (PLCß-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCß-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCß-2 mRNA stability. The 3'UTR of the PLCß-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCß-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCß-2 by LPS was examined using macrophages from mice lacking TTP (TTP-/-). LPS suppressed PLCß-2 expression to the same extent in wild type (WT) and TTP-/- macrophages. Also, the rate of decay of PLCß-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP-/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCß-2 mRNA in macrophages.


Subject(s)
AU Rich Elements/physiology , Macrophages/metabolism , Phospholipase C beta/genetics , RNA Stability/drug effects , Tristetraprolin/physiology , 3' Untranslated Regions/genetics , Animals , Cells, Cultured , Diabetes Mellitus, Experimental , Lipopolysaccharides/pharmacology , Mice , RAW 264.7 Cells , RNA-Binding Proteins
12.
Wiley Interdiscip Rev RNA ; 6(4): 351-67, 2015.
Article in English | MEDLINE | ID: mdl-25832716

ABSTRACT

PTB-associated splicing factor (PSF) is an abundant and essential nucleic acid-binding protein that participates in a wide range of gene regulatory processes and cellular response pathways. At the protein level, PSF consists of multiple domains, many of which remain poorly characterized. Although grouped in a family with the proteins p54nrb/NONO and PSPC1 based on sequence homology, PSF contains additional protein sequence not included in other family members. Consistently, PSF has also been implicated in functions not ascribed to p54nrb/NONO or PSPC1. Here, we provide a review of the cellular activities in which PSF has been implicated and what is known regarding the mechanisms by which PSF functions in each case. We propose that the complex domain arrangement of PSF allows for its diversity of function and integration of activities. Finally, we discuss recent evidence that individual activities of PSF can be regulated independently from one another through the activity of domain-specific co-factors.


Subject(s)
RNA-Binding Proteins/metabolism , Animals , Humans , PTB-Associated Splicing Factor , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry
13.
RNA ; 20(9): 1419-30, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25047043

ABSTRACT

Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H2 is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 can be transiently induced by external stimuli, such as pro-inflammatory cytokines. Interestingly, COX-2 is overexpressed in numerous cancers, including lung cancer. MicroRNAs (miRNAs) are small RNA molecules that function to regulate gene expression. Previous studies have implicated an important role for miRNAs in human cancer. We demonstrate here that miR-146a expression levels are significantly lower in lung cancer cells as compared with normal lung cells. Conversely, lung cancer cells have higher levels of COX-2 protein and mRNA expression. Introduction of miR-146a can specifically ablate COX-2 protein and the biological activity of COX-2 as measured by prostaglandin production. The regulation of COX-2 by miR-146a is mediated through a single miRNA-binding site present in the 3' UTR. Therefore, we propose that decreased miR-146a expression contributes to the up-regulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will give us key insights into how COX-2 expression is involved in the development of a metastatic condition.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Cyclooxygenase 2/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , MicroRNAs/physiology , 3' Untranslated Regions , Base Sequence , Cell Line, Tumor , Gene Expression Profiling , Humans , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Sequence Homology, Nucleic Acid
14.
Methods Mol Biol ; 1125: 157-67, 2014.
Article in English | MEDLINE | ID: mdl-24590788

ABSTRACT

3' end formation of eukaryotic messenger RNAs (mRNAs) is an essential process that influences mRNA stability, turnover, and translation. Polyadenylation is the process by which mRNAs are cleaved at specific sites in response to specific RNA sequence elements and binding of trans-acting protein factors; these cleaved mRNAs subsequently acquire non-templated poly(A) tails at their 3' ends. Alternative polyadenylation occurs when multiple poly(A) signals are present in the primary mRNA transcript, in either the 3' untranslated region (3'UTR) or other sites within the mRNA, resulting in multiple transcript variants of different lengths. We demonstrate here a new method, termed RHAPA (RNase H alternative polyadenylation assay), that employs conventional RT-PCR with gene-specific oligonucleotide hybridization and RNase H cleavage to directly measure and quantify alternatively polyadenylated transcripts. This method gives an absolute quantified expression level of each transcript variant and provides a way to examine poly(A) signal selection in different cell types and under different conditions. Ultimately, it can be used to further examine posttranscriptional regulation of gene expression.


Subject(s)
Poly A/metabolism , Polyadenylation/physiology , 3' Untranslated Regions/genetics , Poly A/chemistry , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
15.
Wiley Interdiscip Rev RNA ; 4(5): 593-605, 2013.
Article in English | MEDLINE | ID: mdl-23956046

ABSTRACT

Arachidonic acid (AA) is converted by enzymes in an important metabolic pathway to produce molecules known collectively as eicosanoids, 20 carbon molecules with significant physiological and pathological functions in the human body. Cyclooxygenase (COX) enzymes work in one arm of the pathway to produce prostaglandins (PGs) and thromboxanes (TXs), while the actions of 5-lipoxygenase (ALOX5 or 5LO) and its associated protein (ALOX5AP or FLAP) work in the other arm of the metabolic pathway to produce leukotrienes (LTs). The expression of the COX and ALOX5 enzymes that convert AA to eicosanoids is highly regulated at the post- or co-transcriptional level by alternative mRNA splicing, alternative mRNA polyadenylation, mRNA stability, and microRNA (miRNA) regulation. This review article will highlight these mechanisms of mRNA modulation.


Subject(s)
Arachidonic Acid/metabolism , Gene Expression Regulation , Metabolic Networks and Pathways/genetics , RNA Processing, Post-Transcriptional , Transcription, Genetic , 5-Lipoxygenase-Activating Proteins/metabolism , Arachidonate 5-Lipoxygenase/metabolism , Eicosanoids/metabolism , Humans , Prostaglandin-Endoperoxide Synthases/metabolism
16.
RNA Biol ; 9(10): 1255-65, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23085579

ABSTRACT

Polyadenylation is a 3' mRNA processing event that contributes to gene expression by affecting stability, export and translation of mRNA. Human polyadenylation signals (PAS) have core and auxiliary elements that bind polyadenylation factors upstream and downstream of the cleavage site. The majority of mRNAs do not have optimal upstream and downstream core elements and therefore auxiliary elements can aid in polyadenylation efficiency. Auxiliary elements have previously been identified and studied in a small number of mRNAs. We previously used a global approach to examine auxiliary elements to identify overrepresented motifs by a bioinformatic survey. This predicted information was used to direct our in vivo validation studies, all of which were accomplished using both a tandem in vivo polyadenylation assay and using reporter protein assays measured as luciferase activity. Novel auxiliary elements were placed in a test polyadenylation signal. An in vivo polyadenylation assay was used to determine the strength of the polyadenylation signal. All but one of the novel auxiliary elements enhanced the test polyadenylation signal. Effects of these novel auxiliary elements were also measured by a luciferase assay when placed in the 3' UTR of a firefly luciferase reporter. Two novel downstream auxiliary elements and all of the novel upstream auxiliary elements showed an increase in reporter protein levels. Many well known auxiliary polyadenylation elements have been found to occur in multiple sets. However, in our study, multiple copies of novel auxiliary elements brought reporter protein levels as well as polyadenylation choice back to wild type levels. Structural features of these novel auxiliary elements may also affect the role of auxiliary elements. A MS2 structure placed upstream of the polyadenylation signal can affect polyadenylation in both the positive and negative direction. A large change in RNA structure by using novel complementary auxiliary element also decreased polyadenylation choice and reporter protein levels. Therefore, we conclude that RNA structure has an important role in polyadenylation efficiency.


Subject(s)
3' Untranslated Regions , Polyadenylation , RNA Processing, Post-Transcriptional , Response Elements , mRNA Cleavage and Polyadenylation Factors/genetics , Base Sequence , Genes, Reporter , HEK293 Cells , HeLa Cells , Humans , Luciferases , Molecular Sequence Data , Nucleic Acid Conformation , mRNA Cleavage and Polyadenylation Factors/metabolism
17.
Comp Funct Genomics ; 2012: 876893, 2012.
Article in English | MEDLINE | ID: mdl-22400011

ABSTRACT

Almost all eukaryotic mRNAs possess 3' ends with a polyadenylate (poly(A)) tail. This poly(A) tail is not encoded in the genome but is added by the process of polyadenylation. Polyadenylation is a two-step process, and this process is accomplished by multisubunit protein factors. Here, we comprehensively compare the protein machinery responsible for polyadenylation of mRNAs across many evolutionary divergent species, and we have found these protein factors to be remarkably conserved in nature. These data suggest that polyadenylation of mRNAs is an ancient process.

18.
Wiley Interdiscip Rev RNA ; 2(1): 22-31, 2011.
Article in English | MEDLINE | ID: mdl-21956967

ABSTRACT

Alternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3(') end formation as well as interactions with other RNA-mediated and RNA processing mechanisms.


Subject(s)
Alternative Splicing/physiology , Gene Expression Regulation , Polyadenylation/physiology , RNA, Messenger/metabolism , Alternative Splicing/genetics , Animals , Gene Expression Regulation/genetics , Humans , Models, Biological , Polyadenylation/genetics , RNA, Messenger/genetics
19.
Arch Virol ; 156(6): 931-8, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21298455

ABSTRACT

The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.


Subject(s)
Protein Interaction Domains and Motifs , Ribonucleoprotein, U1 Small Nuclear/metabolism , Viral Nonstructural Proteins/metabolism , Yellow fever virus , Amino Acid Sequence , Animals , Binding Sites , Chlorocebus aethiops , Conserved Sequence , HeLa Cells , Humans , Immunoprecipitation , Polymerase Chain Reaction , Protein Binding , RNA, Viral , Ribonucleoprotein, U1 Small Nuclear/chemistry , Two-Hybrid System Techniques , Vero Cells , Viral Nonstructural Proteins/chemistry , Yellow fever virus/genetics , Yellow fever virus/metabolism
20.
Wiley Interdiscip Rev RNA ; 2(1): 23-31, 2011.
Article in English | MEDLINE | ID: mdl-21278855

ABSTRACT

Alternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3' end formation, as well as interactions with other RNA-mediated and RNA processing mechanisms.


Subject(s)
Alternative Splicing/physiology , Eukaryota/genetics , Gene Expression Regulation , Polyadenylation/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Alternative Splicing/genetics , Animals , Efficiency , Eukaryota/metabolism , Eukaryotic Cells/metabolism , Humans , Models, Biological , Polyadenylation/genetics
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