[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. WIREs RNA 2013, 4:593–605. doi: 10.1002/wrna.1177
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Conflict of interest: The authors have no conflicts of interest to declare.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. WIREs RNA 2011 2 22–31 DOI: 10.1002/wrna.47
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[Show abstract][Hide abstract] ABSTRACT: The human MeCP2 gene encodes a ubiquitously expressed methyl CpG binding protein. Mutations in this gene cause a neurodevelopmental disorder called Rett Syndrome (RS). Mutations identified in the coding region of MeCP2 account for approximately 65% of all RS cases. However, 35% of all patients do not show mutations in the coding region of MeCP2, suggesting that mutations in non-coding regions likely exist that affect MeCP2 expression rather than protein function. The gene is unusual in that is has a >8.5 kb 3' untranslated region (3' UTR), and the size of the 3'UTR is differentially regulated in various tissues because of distinct polyadenylation signals. We have identified putative cis-acting auxiliary regulatory elements that play a role in alternative polyadenylation of MeCP2 using an in vivo polyadenylation reporter assay and in a luciferase assay. These cis-acting auxiliary elements are found both upstream and downstream of the core CPSF binding sites. Mutation of one of these cis-acting auxiliary elements, a G-rich element (GRS) significantly reduced MeCP2 polyadenylation efficiency in vivo. We further investigated what trans-acting factor(s) might be binding to this cis-acting element and found that hnRNP F protein binds specifically to the element. We next investigated the MeCP2 3' UTRs by performing quantitative real-time PCR; the data suggest that altered RNA stability is not a major factor in differential MeCP2 3' UTR usage. In sum, the mechanism(s) of regulated alternative 3'UTR usage of MeCP2 are complex, and insight into these mechanisms will aid our understanding of the factors that influence MeCP2 expression.
[Show abstract][Hide abstract] ABSTRACT: Regulation of gene expression by RNA processing mechanisms is now understood to be an important level of control in mammalian cells. Regulation at the level of RNA transcription, splicing, polyadenylation, nucleo-cytoplasmic transport, and translation into polypeptides has been well-studied. Alternative RNA processing events, such as alternative splicing, also have been recognized as key contributors to the complexity of mammalian gene expression. Pre-messenger RNAs (pre-mRNAs) may be polyadenylated in several different ways due to more than one polyadenylation signal, allowing a single gene to encode multiple mRNA transcripts. However, alternative polyadenylation has only recently taken the field as a major player in gene regulation. This review summarizes what is currently known about alternative polyadenylation. It covers results from bioinformatics, as well as those from investigations of viral and tissue-specific studies and, importantly, will set the stage for what is yet to come.
ACS Chemical Biology 10/2008; 3(10):609-17. DOI:10.1021/cb800138w · 5.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian gene expression can be regulated through various post-transcriptional events, including altered mRNA stability, translational control, and RNA-processing events such as 3'-end formation or polyadenylation (pA). It has become clear in recent years that pA is governed by several core sequence elements and often regulated by additional auxiliary sequence elements. These regulatory events are frequently not reproducible in in vitro assays. Therefore, in vivo methods to measure mRNA pA were developed to meet this need and are described here.
[Show abstract][Hide abstract] ABSTRACT: Two cyclooxygenase (COX) enzymes, COX-1 and COX-2, are present in human cells. While COX-1 is constitutively expressed, COX-2 is inducible and up-regulated in response to many signals. Since increased transcriptional activity accounts for only part of COX-2 up-regulation, we chose to explore other RNA processing mechanisms in the regulation of this gene. Previously, we showed that COX-2 is regulated by alternative polyadenylation, and that the COX-2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs) that are very important in efficient polyadenylation. To explore trans-acting protein factors interacting with these cis-acting RNA elements, we performed pull-down assays with HeLa nuclear extract and biotinylated RNA oligonucleotides representing COX-2 USEs. We identified PSF, p54(nrb), PTB, and U1A as proteins specifically bound to the COX-2 USEs. We further explored their participation in polyadenylation using MS2 phage coat protein-MS2 RNA binding site tethering assays, and found that tethering any of these four proteins to the COX-2 USE mutant RNA can compensate for these cis-acting elements. Finally, we suggest that these proteins (p54(nrb), PTB, PSF, and U1A) may interact as a complex since immunoprecipitations of the transfected MS2 fusion proteins coprecipitate the other proteins.
[Show abstract][Hide abstract] ABSTRACT: Human CstF-77 is one of the three subunits of cleavage stimulation factor (CstF) that is essential for mRNA polyadenylation. Its Drosophila homologue, suppressor of forked [su(f)], contains an intronic poly(A) site, which can lead to a short transcript without a stop codon. By both bioinformatic searches and validation with molecular biology experiments, we found that human and mouse CstF-77 genes also contain an intronic poly(A) site, which can be utilized to produce short CstF-77 transcripts lacking sequences encoding domains that are involved in many of the CstF-77 functions. The genomic sequence surrounding the poly(A) site is highly conserved among all vertebrates, but is not present in non-vertebrate species. Using public Serial Analysis of Gene Expression (SAGE) data, we found that the intronic poly(A) site is utilized in a wide range of tissues. This finding indicates that vertebrates may employ a similar alternative polyadenylation mechanism to modulate CstF-77, highlighting the importance of the regulation of CstF-77 in various species.
[Show abstract][Hide abstract] ABSTRACT: The U1 snRNP-A (U1A) protein has been known for many years as a component of the U1 snRNP. We have previously described a form of U1A present in human cells in significant amounts that is not associated with the U1 snRNP or U1 RNA but instead is part of a novel complex of non-snRNP proteins that we have termed snRNP-free U1A, or SF-A. Antibodies that specifically recognize this complex inhibit in vitro splicing and polyadenylation of pre-mRNA, suggesting that this complex may play an important functional role in these mRNA-processing activities. This finding was underscored by the determination that one of the components of this complex is the polypyrimidine-tract-binding protein-associated splicing factor, PSF. In order to further our studies on this complex and to determine the rest of the components of the SF-A complex, we prepared several stable HeLa cell lines that overexpress a tandem-affinity-purification-tagged version of U1A (TAP-tagged U1A). Nuclear extract was prepared from one of these cell lines, line 107, and affinity purification was performed along with RNase treatment. We have used mass spectrometry analysis to identify the candidate factors that associate with U1A. We have now identified and characterized PSF, p54(nrb), and p68 as novel components of the SF-A complex. We have explored the function of this complex in RNA processing, specifically cleavage and polyadenylation, by performing immunodepletions followed by reconstitution experiments, and have found that p54(nrb) is critical.
[Show abstract][Hide abstract] ABSTRACT: Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A1, A2A, A2B, and A3. Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich 3'-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3'-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3'-UTR of TNF-alpha mRNA. The IL-10 3'-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3'-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A(2B) receptor, because the order of potency of selective agonists was 5'-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) = 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A2B antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.
The Journal of Immunology 01/2006; 175(12):8260-70. DOI:10.4049/jimmunol.175.12.8260 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyadenylation is an essential step for the maturation of almost all cellular mRNAs in eukaryotes. In human cells, most poly(A) sites are flanked by the upstream AAUAAA hexamer or a close variant, and downstream U/GU-rich elements. In yeast and plants, additional cis elements have been found to be located upstream of the poly(A) site, including UGUA, UAUA, and U-rich elements. In this study, we have developed a computer program named PROBE (Polyadenylation-Related Oligonucleotide Bidimensional Enrichment) to identify cis elements that may play regulatory roles in mRNA polyadenylation. By comparing human genomic sequences surrounding frequently used poly(A) sites with those surrounding less frequently used ones, we found that cis elements occurring in yeast and plants also exist in human poly(A) regions, including the upstream U-rich elements, and UAUA and UGUA elements. In addition, several novel elements were found to be associated with human poly(A) sites, including several G-rich elements. Thus, we suggest that many cis elements are evolutionarily conserved among eukaryotes, and human poly(A) sites have an additional set of cis elements that may be involved in the regulation of mRNA polyadenylation.
[Show abstract][Hide abstract] ABSTRACT: A biologically important human gene, cyclooxygenase-2 (COX-2), has been proposed to be regulated at many levels. While COX-1 is constitutively expressed in cells, COX-2 is inducible and is upregulated in response to many signals. Since increased transcriptional activity accounts for only part of the upregulation of COX-2, we chose to explore other RNA processing mechanisms in the regulation of this gene. We performed a comprehensive bioinformatics survey, the first of its kind known for human COX-2, which revealed that the human COX-2 gene has alternative polyadenylation (proximal and distal sites) and suggested that use of the alternative polyadenylation signals has tissue specificity. We experimentally established this in HepG2 and HT29 cells. We used an in vivo polyadenylation assay to examine the relative strength of the COX-2 proximal and distal polyadenylation signals, and have shown that the proximal polyadenylation signal is much weaker than the distal one. The efficiency of utilization of many suboptimal mammalian polyadenylation signals is affected by sequence elements located upstream of the AAUAAA, known as upstream efficiency elements (USEs). Here, we used in vivo polyadenylation assays in multiple cell lines to demonstrate that the COX-2 proximal polyadenylation signal contains USEs, mutation of the USEs substantially decreased usage of the proximal signal, and that USE spacing relative to the polyadenylation signal was significant. In addition, mutation of the COX-2 proximal polyadenylation signal to a more optimal sequence enhanced polyadenylation efficiency 3.5-fold. Our data suggest for the first time that alternative polyadenylation of COX-2 is an important post-transcriptional regulatory event.
Nucleic Acids Research 02/2005; 33(8):2565-79. DOI:10.1093/nar/gki544 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: mRNA polyadenylation is a critical cellular process in eukaryotes. It involves 3' end cleavage of nascent mRNAs and addition of the poly(A) tail, which plays important roles in many aspects of the cellular metabolism of mRNA. The process is controlled by various cis-acting elements surrounding the cleavage site, and their binding factors. In this study, we surveyed genome regions containing cleavage sites [herein called poly(A) sites], for 13,942 human and 11,155 mouse genes. We found that a great proportion of human and mouse genes have alternative polyadenylation ( approximately 54 and 32%, respectively). The conservation of alternative polyadenylation type or polyadenylation configuration between human and mouse orthologs is statistically significant, indicating that alternative polyadenylation is widely employed by these two species to produce alternative gene transcripts. Genes belonging to several functional groups, indicated by their Gene Ontology annotations, are biased with respect to polyadenylation configuration. Many poly(A) sites harbor multiple cleavage sites (51.25% human and 46.97% mouse sites), leading to heterogeneous 3' end formation for transcripts. This implies that the cleavage process of polyadenylation is largely imprecise. Different types of poly(A) sites, with regard to their relative locations in a gene, are found to have distinct nucleotide composition in surrounding genomic regions. This large-scale study provides important insights into the mechanism of polyadenylation in mammalian species and represents a genomic view of the regulation of gene expression by alternative polyadenylation.
Nucleic Acids Research 02/2005; 33(1):201-12. DOI:10.1093/nar/gki158 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A subset of lupus patients with severe nephritis and anti-nRNP reactivity produces autoantibodies primarily against two major epitopes of the nRNP A (also known as U1A) protein. These sequences span amino acids 44-56 (A3) and amino acids 103-115 (A6). These two epitopes represent structurally different regions of the protein, as both epitopes are located on the surface, but the A6 epitope is functionally masked in vivo by binding between nRNP A and the U1 RNA. Rabbits were immunized with either the A3 or A6 peptides constructed on a branching polylysine backbone. Rabbits immunized with each of these peptides first developed antibodies directed against the peptide of immunization. With boosting, the immune response of rabbits immunized with the A3 peptide spread to other common antigenic regions of nRNP A. These regions of nRNP A bound by A3 immunized rabbits are very similar to common epitopes in human systemic lupus erythematosus. These A3 immunized rabbits also develop antibodies to common antigenic regions of nRNP 70K, nRNP C, Sm B/B', and Sm D1 proteins, as well as clinical symptoms of systemic lupus erythematosus such as leukopenia and renal insufficiency. On the other hand, rabbits immunized with the A6 peptide only develop antibodies to the peptide of immunization. Anti-A3, but not anti-A6, antibodies are capable of immunoprecipitating native small nuclear ribonucleoprotein complexes. Immunization with the A3 peptide of nRNP A (a surface epitope), but not the A6 peptide (masked), induces an extensive, varied immune response against multiple small nuclear ribonucleoprotein autoantigens similar to that seen in human systemic lupus erythematosus.
Proceedings of the National Academy of Sciences 04/2004; 101(10):3551-6. DOI:10.1073/pnas.0306267101 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The U1A protein can be found both in a small-ribonucleoprotein particle (snRNP) that contains U1 RNA, or in a distinctive fraction, free of the snRNP, the SF-A complex. Both components have been shown to influence post- or co-transcriptional RNA processing reactions in HeLa cells. Since U1A may influence the processing of the immunoglobulin heavy chain pre-mRNA in B-cells, we wanted to see if the levels of U1A in either of its two forms changed following IL-6 stimulation to IgM secretion. Using antibodies that specifically recognize the two forms of U1A, snRNP-associated and snRNP-free, we found that approximately 16% of U1A is in the SF-A form in B-cells. We measured the levels of U1A protein in its two states in human B-cell lines both by flow cytometry and exhaustive immunoprecipitations. We found a significant decrease in the amount of snRNP-associated U1A following cytokine stimulation that correlates with the change-over to the secretory-specific poly(A) site use in the SKW 6.4 cell line. Meanwhile, the number of U1A molecules in the SF-A fraction of the pool remains nearly constant following induction to secretion. Our results suggest that the changing level of U1A in the snRNP fraction may be important for influencing Ig heavy chain mRNA processing.
[Show abstract][Hide abstract] ABSTRACT: We have previously described a novel complex of proteins which contains the U1snRNP-A protein (U1A) but no other small nuclear ribonucleoprotein particle (snRNP) components (O'Connor et al., RNA 1997;3:1444-55). Antibodies to this novel complex inhibit both splicing and polyadenylation in vitro of a test pre-mRNA (O'Connor et al., RNA 1997;3:1444-55; Lutz et al., RNA 1998;4:1493-9). This novel complex of proteins was identified using an unusual mouse monoclonal antibody (MoAb), called MAb 12E12. We were interested to know if autoimmune patient sera were similar to this MoAb. We have discovered a novel specificity of systemic lupus erythematosus patient sera reminiscent of MAb 12E12 in that the patient serum, like 12E12, (1) does not recognize U1A when bound to U1 RNA, (2) recognizes primarily the epitopes in the amino-terminal third of the protein, including RNA recognition motif 1 (RRM1) and (3) inhibits in vitro polyadenylation. These findings may lead to the discovery of previously undescribed autoantigens as components of the novel protein complex, and may provide insight into autoimmune diseases.
[Show abstract][Hide abstract] ABSTRACT: 3'-Untranslated regions (UTRs) of genes often contain key regulatory elements involved in gene expression control. A high degree of evolutionary conservation in regions of the 3'-UTR suggests important, conserved elements. In particular, we are interested in those elements involved in regulation of 3' end formation. In addition to canonical sequence elements, auxiliary sequences likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3' end formation. Close inspection of the COL1A2 3'-UTR reveals an unusual feature of two closely spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs are important auxiliary polyadenylation elements in mammalian genes.