ABSTRACT
p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.
Subject(s)
Alternative Splicing/genetics , Hyaluronan Receptors/genetics , RNA Splicing/genetics , RNA-Binding Proteins/metabolism , Carcinogenesis/genetics , Colorectal Neoplasms/genetics , Gene Knockdown Techniques , Gene Silencing , HCT116 Cells , HEK293 Cells , Humans , Hyaluronan Receptors/metabolism , Protein Binding/genetics , RNA Precursors/metabolism , RNA-Binding Proteins/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.
Subject(s)
Benzomorphans , Calcium , Animals , Mice , Hydrogen Peroxide , Sigma-1 Receptor , Thapsigargin , Calcium SignalingABSTRACT
In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1's interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1's binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.
Subject(s)
G-Quadruplexes , Alternative Splicing , Base Sequence , Oncogenes , RNA/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.
Subject(s)
Cell Proliferation/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Heterogeneous Nuclear Ribonucleoprotein D0/metabolism , RNA, Circular/metabolism , 3' Untranslated Regions , Binding Sites , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/metabolism , HeLa Cells , Humans , RNA, Circular/chemistry , RNA, Messenger/metabolismABSTRACT
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse cellular processes. Although the vast majority of lncRNAs are expressed at lower levels compared to messenger RNAs (mRNAs), many lncRNAs play a central role in the regulation of cellular homeostasis and gene expression. With the advancement of next generation sequencing technologies, recent studies illustrate the diversity of lncRNA function. This diversity can be due to differences in their mechanisms of action, spatio-temporal expression, and/or abundance, all of which can vary depending on the particular cell type or tissue. Here, we discuss how the abundance of lncRNAs is an important feature that is often linked to their functions, and why it is crucial to quantitate lncRNA abundance, its local concentration within a cell or a tissue or the dynamic changes in expression levels during cell cycle progression or upon environmental stimuli, to shed light on their physiological roles.
Subject(s)
RNA, Long Noncoding , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/geneticsABSTRACT
Using RNA sequencing (RNA-Seq), we compared the expression patterns of circular RNAs in proliferating (early-passage) and senescent (late-passage) human diploid WI-38 fibroblasts. Among the differentially expressed senescence-associated circRNAs (which we termed 'SAC-RNAs'), we identified CircPVT1, generated by circularization of an exon of the PVT1 gene, as a circular RNA showing markedly reduced levels in senescent fibroblasts. Reducing CircPVT1 levels in proliferating fibroblasts triggered senescence, as determined by a rise in senescence-associated ß-galactosidase activity, higher abundance of CDKN1A/P21 and TP53, and reduced cell proliferation. Although several microRNAs were predicted to bind CircPVT1, only let-7 was found enriched after pulldown of endogenous CircPVT1, suggesting that CircPVT1 might selectively modulate let-7 activity and hence expression of let-7-regulated mRNAs. Reporter analysis revealed that CircPVT1 decreased the cellular pool of available let-7, and antagonizing endogenous let-7 triggered cell proliferation. Importantly, silencing CircPVT1 promoted cell senescence and reversed the proliferative phenotype observed after let-7 function was impaired. Consequently, the levels of several proliferative proteins that prevent senescence, such as IGF2BP1, KRAS and HMGA2, encoded by let-7 target mRNAs, were reduced by silencing CircPVT1. Our findings indicate that the SAC-RNA CircPVT1, elevated in dividing cells and reduced in senescent cells, sequesters let-7 to enable a proliferative phenotype.
Subject(s)
Cellular Senescence/genetics , RNA, Long Noncoding/metabolism , RNA/metabolism , Cell Line , Cell Proliferation , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , RNA, CircularABSTRACT
High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.
Subject(s)
Exons , High-Throughput Nucleotide Sequencing , Introns , Poly A/genetics , RNA, Messenger/chemistry , RNA/isolation & purification , Animals , Base Sequence , Cell Line , Computational Biology , Exoribonucleases/chemistry , HeLa Cells , Humans , Mice , Molecular Sequence Annotation , Myoblasts/cytology , Myoblasts/metabolism , Poly A/metabolism , Polyadenylation , RNA/genetics , RNA/metabolism , RNA Cleavage , RNA, Circular , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The function of the vast majority of mammalian long noncoding (lnc) RNAs remains unknown. Here, analysis of a highly abundant mammalian lncRNA, OIP5-AS1, known as cyrano in zebrafish, revealed that OIP5-AS1 reduces cell proliferation. In human cervical carcinoma HeLa cells, the RNA-binding protein HuR, which enhances cell proliferation, associated with OIP5-AS1 and stabilized it. Tagging OIP5-AS1 with MS2 hairpins to identify associated microRNAs revealed that miR-424 interacted with OIP5-AS1 and competed with HuR for binding to OIP5-AS1. We further identified a 'sponge' function for OIP5-AS1, as high levels of OIP5-AS1 increased HuR-OIP5-AS1 complexes and prevented HuR interaction with target mRNAs, including those that encoded proliferative proteins, while conversely, lowering OIP5-AS1 increased the abundance of HuR complexes with target mRNAs. We propose that OIP5-AS1 serves as a sponge or a competing endogenous (ce)RNA for HuR, restricting its availability to HuR target mRNAs and thereby repressing HuR-elicited proliferative phenotypes.
Subject(s)
ELAV-Like Protein 1/genetics , Gene Expression Regulation , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , ADAM Proteins/genetics , ADAM Proteins/metabolism , Base Sequence , Binding Sites , Binding, Competitive , Cell Proliferation/genetics , ELAV-Like Protein 1/antagonists & inhibitors , ELAV-Like Protein 1/metabolism , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/metabolism , Molecular Sequence Data , Protein Binding , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.
Subject(s)
Cyclin D1/genetics , Muscle Development/genetics , Myogenic Regulatory Factor 5/genetics , RNA, Messenger/genetics , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Cyclin D1/metabolism , Gene Expression Profiling , Gene Expression Regulation , Mice , Microarray Analysis , Myoblasts , Myogenic Regulatory Factor 5/metabolism , Protein Binding , Protein Biosynthesis , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
HuR influences gene expression programs and hence cellular phenotypes by binding to hundreds of coding and noncoding linear RNAs. However, whether HuR binds to circular RNAs (circRNAs) and impacts on their function is unknown. Here, we have identified en masse circRNAs binding HuR in human cervical carcinoma HeLa cells. One of the most prominent HuR target circRNAs was hsa_circ_0031288, renamed CircPABPN1 as it arises from the PABPN1 pre-mRNA. Further analysis revealed that HuR did not influence CircPABPN1 abundance; interestingly, however, high levels of CircPABPN1 suppressed HuR binding to PABPN1 mRNA. Evaluation of PABPN1 mRNA polysomes indicated that PABPN1 translation was modulated positively by HuR and hence negatively by CircPABPN1. We propose that the extensive binding of CircPABPN1 to HuR prevents HuR binding to PABPN1 mRNA and lowers PABPN1 translation, providing the first example of competition between a circRNA and its cognate mRNA for an RBP that affects translation.
Subject(s)
ELAV-Like Protein 1/metabolism , Gene Expression Regulation , Poly(A)-Binding Protein I/genetics , Protein Biosynthesis , RNA/genetics , RNA/metabolism , Base Sequence , Binding Sites , Cell Line, Tumor , Humans , Models, Biological , Protein Binding , RNA, Circular , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
At the beginning of the 19th century, gynaecological cancer, mainly cancer of the uterus and cervix was a dreadful, incurable affection. However, the popularization of the three fundamentals in surgery, anesthesia, asepsis and haemostasis, ushered the golden age of operative gynaecology. During that period distinguished surgeons/gynaecologists such as Friedrich Benjamin Osiander (1759-1822), Elias von Siebold (1775-1828) and Joseph-Claude-Anthelme Récamier (1774-1852) contributed to the development of the operative techniques, providing a therapeutic solution in gynaecological cancer.
Subject(s)
Genital Neoplasms, Female/history , Gynecologic Surgical Procedures/history , Female , Genital Neoplasms, Female/surgery , History, 19th Century , History, 20th Century , HumansABSTRACT
Circular RNAs (circRNAs) are widely expressed in animal cells, but their biogenesis and functions are poorly understood. CircRNAs have been shown to act as sponges for miRNAs and may also potentially sponge RNA-binding proteins (RBPs) and are thus predicted to function as robust posttranscriptional regulators of gene expression. The joint analysis of large-scale transcriptome data coupled with computational analyses represents a powerful approach to elucidate possible biological roles of ribonucleoprotein (RNP) complexes. Here, we present a new web tool, CircInteractome (circRNA interactome), for mapping RBP- and miRNA-binding sites on human circRNAs. CircInteractome searches public circRNA, miRNA, and RBP databases to provide bioinformatic analyses of binding sites on circRNAs and additionally analyzes miRNA and RBP sites on junction and junction-flanking sequences. CircInteractome also allows the user the ability to (1) identify potential circRNAs which can act as RBP sponges, (2) design junction-spanning primers for specific detection of circRNAs of interest, (3) design siRNAs for circRNA silencing, and (4) identify potential internal ribosomal entry sites (IRES). In sum, the web tool CircInteractome, freely accessible at http://circinteractome.nia.nih.gov, facilitates the analysis of circRNAs and circRNP biology.
Subject(s)
Computational Biology/methods , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , RNA/chemistry , RNA/metabolism , Binding Sites , Databases, Genetic , Humans , Models, Molecular , RNA, Circular , Sequence Analysis, RNA , Web BrowserABSTRACT
Noncoding RNAs (ncRNAs) and RNA-binding proteins are potent post-transcriptional regulators of gene expression. The ncRNA 7SL is upregulated in cancer cells, but its impact upon the phenotype of cancer cells is unknown. Here, we present evidence that 7SL forms a partial hybrid with the 3'-untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. We propose that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53. Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.
Subject(s)
ELAV Proteins/metabolism , Gene Expression Regulation, Neoplastic , Protein Biosynthesis , RNA, Small Cytoplasmic/metabolism , Signal Recognition Particle/metabolism , Tumor Suppressor Protein p53/genetics , 3' Untranslated Regions , Autophagy , Binding, Competitive , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cellular Senescence , HeLa Cells , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , RNA, Messenger/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
We aim to present the first case of a pregnancy achieved by administering sildenafil (Viagra) to a woman not responding to controlled ovarian hyperstimulation (COH) with the sole use of gonadotropins. A 37-year-old woman underwent COH, as part of an intracytoplasmic sperm injection (ICSI) cycle, with the combination of r-FSH and HMG for 13 d, without evidence of follicular growth. The addition of oral sildenafil at a dose of 50 mg per day for a total of five doses improved the ovarian response and resulted in the retrieval of 10 oocytes. Three embryos were transferred to the uterine cavity resulting in a successful pregnancy and, eventually, the delivery of a healthy neonate. Conclusively, the use of sildenafil as an adjunct to COH protocols may enhance ovarian response in a woman with poor ovarian response (POR) and merits further research.
Subject(s)
Ovarian Follicle/drug effects , Ovulation Induction/methods , Piperazines/administration & dosage , Sulfonamides/administration & dosage , Vasodilator Agents/administration & dosage , Adult , Female , Humans , Infant, Newborn , Male , Pregnancy , Purines/administration & dosage , Sildenafil Citrate , Sperm Injections, Intracytoplasmic/methodsABSTRACT
Mineral gypsum (selenite) stones have been used extensively by ancient Cretans in the Minoan Palace of Knossos (Crete, Greece), mostly for building and ornamental purposes. Exposure of mineral gypsum to environmental stresses (temperature fluctuations, rain, air-borne pollutants, soluble salts, etc.) causes solubility-driven degradation, and loss of cohesion of the crystal aggregates, with ensuing aesthetic degradation. In this work, the efficiency of four consolidants for artificial gypsum specimens is presented and evaluated based on drilling resistance measurements [drilling resistance measuring system (DRMS)]. Two of them (commercial names RC-70 and RC-90, RC = Rhodorsil Consolidante) are alkoxysilane-based and they are considered as benchmark consolidants. The other two [3-(trihydroxysilyl)propyl methylphosphonate monosodium salt, TRIMEPHONA, and 3-(trihydroxysilyl)propylamino-diphosphonate, TRIPADIPHOS] are multifunctional consolidants because they possess a self-condensable (after hydrolysis) trihydroxysilyl [-Si(OH)3] moiety and phosphonate groups (one in the former, two in the latter). Consolidants RC-70 and RC-90 exhibit rather low consolidation effectiveness. This is not unexpected, as these are alkoxysilane-based and act simply as "fillers" for the pores of the gypsum. Consolidant TRIMEPHONA demonstrates an enhanced level of consolidation action. This is due to its double functionality, i.e., the presence of an anionic phosphorus-based moiety that anchors onto the gypsum surface, and a condensable silane triol [-Si(OH)3] unit. Consolidant TRIPADIPHOS shows excellent gypsum consolidation features and is much more efficient (per unit concentration) than all other tested consolidants. This is assigned to its better gypsum anchoring ability via surface Ca-complexation. Selected compressive strength studies were performed on gypsum samples treated with the phosphorus-based consolidants, and corroborate the findings from DRMS. To shed further light on possible binding modes of the phosphonate moiety on surface Ca2+ sites in gypsum, two model compounds were synthesized and structurally characterized, Ca-C2D and Ca-C3D (C2D = ethylamino-di(methylenephosphonic acid) and C3D = propylamino-di(methylenephosphonic acid).
ABSTRACT
Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.
Subject(s)
Colorectal Neoplasms , Transcriptome , Male , Humans , Animals , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/metabolism , Gene Expression Regulation , Colorectal Neoplasms/metabolism , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Cytoskeletal Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Muscleblind-like 1 (MBNL1) is a splicing regulator that controls developmentally regulated alternative splicing of a large number of exons including exon 11 of the Insulin Receptor (IR) gene and exon 5 of the cardiac Troponin T (cTNT) gene. There are three paralogs of MBNL in humans, all of which promote IR exon 11 inclusion and cTNT exon 5 skipping. Here, we identify a cluster of three binding sequences located downstream of IR exon 11 that constitute the MBNL1 response element and a weaker response element in the upstream intron. In addition, we used sequential deletions to define the functional domains of MBNL1 and MBNL3. We demonstrate that the regions required for splicing regulation are separate from the two pairs of zinc-finger RNA-binding domains. MBNL1 and MBNL3 contain core regulatory regions for both activation and repression located within an 80-amino-acid segment located downstream of the N-terminal zinc-finger pair. Deletions of these regions abolished regulation without preventing RNA binding. These domains have common features with the CUG-BP and ETR3-like Factor (CELF) family of splicing regulators. These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins.
Subject(s)
Alternative Splicing , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Exons , Humans , Molecular Sequence Data , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Regulatory Sequences, Ribonucleic Acid , Sequence DeletionABSTRACT
MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. In mammalian cells, miRNAs typically suppress mRNA stability and/or translation through partial complementarity with target mRNAs. Each miRNA can regulate a wide range of mRNAs, and a single mRNA can be regulated by multiple miRNAs. Through these complex regulatory interactions, miRNAs participate in many cellular processes, including carcinogenesis. By altering gene expression patterns, cancer cells can develop specific phenotypes that allow them to proliferate, survive, secure oxygen and nutrients, evade immune recognition, invade other tissues and metastasize. At the same time, cancer cells acquire miRNA signature patterns distinct from those of normal cells; the differentially expressed miRNAs contribute to enabling the cancer traits. Over the past decade, several miRNAs have been identified, which functioned as oncogenic miRNAs (oncomiRs) or tumor-suppressive miRNAs (TS-miRNAs). In this review, we focus specifically on TS-miRNAs and their effects on well-established cancer traits. We also discuss the rising interest in TS-miRNAs in cancer therapy.
Subject(s)
Genes, Tumor Suppressor , MicroRNAs , Neoplasms , Quantitative Trait Loci , RNA, Neoplasm , Animals , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasms/genetics , Neoplasms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolismABSTRACT
Insulin system including ligands (insulin and IGFs) and their shared receptors (IR and IGFR) are critical regulators of insulin signaling and glucose homeostasis. Altered insulin system is associated with major pathological conditions like diabetes and cancer. The mRNAs encoding for these ligands and their receptors are posttranscriptionally controlled by three major groups of regulators; (i) alternative splicing regulatory factors; (ii) turnover and translation regulator RNA-binding proteins (TTR-RBPs); and (iii) non-coding RNAs including miRNAs and long non-coding RNAs (lncRNAs). In this review, we discuss the influence of these regulators on alternative splicing, mRNA stability and translation. Due to the pathological impacts of insulin system, we also discussed the possibilities of discovering new potential regulators which will improve understanding of insulin system and associated diseases.
Subject(s)
Insulin/metabolism , Ligands , Receptors, Somatomedin/metabolism , Somatomedins/metabolism , Alternative Splicing , Humans , Insulin/genetics , Protein Processing, Post-Translational , RNA, Messenger/metabolism , RNA, Untranslated/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Receptors, Somatomedin/genetics , Somatomedins/geneticsABSTRACT
BACKGROUND AND AIM: Unexplained recurrent pregnancy loss has been a a challenging research task to experts since there is no explicit pathophysiological mechanism and therefore, the treatment remains elusive. Immunological imbalance and morphological abnormalities are under investigation. This study aims to evaluate the implication of MMP-2, MMP-9, EGFR, and IL-8 in recurrent pregnancy loss cases. MATERIALS & METHODS: The study was carried out through comparison among two groups; the unexplained miscarriage group which consisted of 22 women, and the control group consisted of 18 women, who had electively terminated their pregnancies. Both groups were in the first trimester of gestation. The specimens included the trophoblast, decidua basalis, and decidua parietalis. The study was conducted via immunohistochemical methods. Antibodies were used against MMP-2, MMP-9, EGFR, and IL-8. The results were presented at a contingency table and were statistically analyzed with the Chi-Square Test (X2). RESULTS: There were remarkable disparities in some cases in the comparison of the two groups. MMP-9 was detected significantly high in recurrent pregnancy loss (RPL) cases, both on trophoblastic and decidual specimens (p-value < .00001), MMP-2 displayed no difference among the two groups (mild to moderate detection on trophoblast and almost negative on decidual tissues). EGFR was highly detected in trophoblastic tissue (p-value = .014). IL-8 detection was particularly different in both trophoblast and decidua parietalis of the two groups (p-value < .01). CONCLUSION: The study revealed both morphological and immunological dysregulations that might participate in the RPL pathogenesis.