RESUMO
Cytoplasmic streaming with extremely high velocity (â¼70 µm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 µm s-1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 µm s-1, one-third of the cytoplasmic streaming velocity in Chara Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 µm s-1 These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.
Assuntos
Chara/genética , Corrente Citoplasmática/fisiologia , Miosinas/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Corrente Citoplasmática/genética , Miosinas/genéticaRESUMO
Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.
Assuntos
Arabidopsis , Calmodulina , Calmodulina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ligação ProteicaRESUMO
Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 µm s-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.
Assuntos
Arabidopsis/genética , Marchantia/genética , Miosinas/genética , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente ModificadasRESUMO
Plant myosin XI acts as a motive force for cytoplasmic streaming through interacting with actin filaments within the cell. Arabidopsis thaliana (At) has 13 genes belonging to the myosin XI family. Previous reverse genetic approaches suggest that At myosin XIs are partially redundant, but are functionally diverse for their specific tasks within the plant. However, the tissue-specific expression and enzymatic properties of myosin XIs have to date been poorly understood, primarily because of the difficulty in cloning and expressing large myosin XI genes and proteins. In this study, we cloned full-length cDNAs and promoter regions for all 13 At myosin XIs and identified tissue-specific expression (using promoter-reporter assays) and motile and enzymatic activities (using in vitro assays). In general, myosins belonging to the same class have similar velocities and ATPase activities. However, the velocities and ATPase activities of the 13 At myosin XIs are significantly different and are classified broadly into three groups based on velocity (high group, medium group and low group). Interestingly, the velocity groups appear roughly correlated with the tissue-specific expression patterns. Generally, ubiquitously expressed At myosin XIs belong to the medium-velocity group, pollen-specific At myosin XIs belong to the high-velocity group and only one At myosin XI (XI-I) is classified as belonging to the low-velocity group. In this study, we demonstrated the diversity of the 13 myosin XIs in Arabidopsis at the molecular and tissue levels. Our results indicate that myosin XIs in higher plants have distinct motile and enzymatic activities adapted for their specific roles.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Miosinas/metabolismo , Adenosina Trifosfatases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Genes de Plantas/genética , Glucuronidase/metabolismo , Miosinas/genética , Regiões Promotoras Genéticas/genéticaRESUMO
There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.
Assuntos
Actinas/química , Proteínas de Arabidopsis/química , Proteínas Motores Moleculares/química , Movimento (Física) , Miosinas/química , Actinas/ultraestrutura , Proteínas de Arabidopsis/ultraestrutura , Ativação Enzimática , Proteínas Motores Moleculares/ultraestrutura , Miosinas/ultraestrutura , Ligação ProteicaRESUMO
Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Motores Moleculares/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Corrente Citoplasmática , Genes Reporter , Proteínas Motores Moleculares/genética , Organelas/metabolismo , Transporte ProteicoRESUMO
Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 µM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 µm/s, respectively, from which the value for full-length ATM1 is calculated to be â¼0.2 µm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was â¼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dinitrobenzenos/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Microscopia Confocal , Miosinas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Protoplastos/citologia , Protoplastos/metabolismo , Sulfanilamidas/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of 'Tough Decoy (TuD) RNA' molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2'-O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.
Assuntos
MicroRNAs/antagonistas & inibidores , RNA/química , Sítios de Ligação , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , MicroRNAs/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/metabolismo , TransfecçãoRESUMO
All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head-tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin-myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head-tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.
Assuntos
Miosinas de Músculo Liso/metabolismo , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Galinhas , Sequência Conservada , Leucina/genética , Leucina/metabolismo , Mutação , Prolina/genética , Prolina/metabolismo , Miosinas de Músculo Liso/genéticaRESUMO
The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3'UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9, miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3'UTR to its transcript. Importantly, simultaneous mutations of both the miR-9- and miR-16-binding sites in the CDX2 3'UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family.
Assuntos
Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , MicroRNAs/fisiologia , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , Neoplasias Colorretais , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genéticaRESUMO
Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.
Assuntos
Citoesqueleto de Actina , Drosophila melanogaster , Animais , Rotação , Drosophila melanogaster/metabolismo , Citoesqueleto de Actina/metabolismo , Miosina Tipo I/metabolismo , Membrana Celular/metabolismo , Actinas/metabolismoRESUMO
Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3'-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.
Assuntos
Glicoproteínas/metabolismo , MicroRNAs/genética , Vírus Oncolíticos/fisiologia , Vaccinia virus/fisiologia , Regiões 3' não Traduzidas/genética , Animais , Células CACO-2 , Linhagem Celular Tumoral , Feminino , Glicoproteínas/genética , Células HeLa , Humanos , Camundongos , Camundongos SCID , Vírus Oncolíticos/genética , Vaccinia virus/genética , Replicação Viral/genética , Replicação Viral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: - 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2-3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.
Assuntos
Arabidopsis/metabolismo , Corrente Citoplasmática , Cadeias Pesadas de Miosina/metabolismo , Organelas/metabolismo , Arabidopsis/genética , Cadeias Pesadas de Miosina/genética , Organelas/genéticaRESUMO
The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Fator de Transcrição RelB/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Linfotoxina-alfa/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.
Assuntos
MicroRNAs/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/química , Sítios de Ligação , Linhagem Celular , Genes Reporter , Vetores Genéticos , Humanos , Lentivirus/genética , MicroRNAs/análise , MicroRNAs/antagonistas & inibidores , Conformação de Ácido Nucleico , RNA não Traduzido/metabolismo , Transdução GenéticaRESUMO
In our recent study showing a correlation between Brm-deficiency and undifferentiated status of gastric cancer, we found that the Brm-type SWI/SNF complex is required for villin expression. To elucidate intestinal villin regulation more precisely, we here analyzed structure and function of the promoter of human villin. About 1.1 kb upstream of the determined major transcription start site, we identified a highly conserved region (HCR-Cdx) among mammals, which contains two binding sites for Cdx. Expression analyses of 30 human gastrointestinal cell lines suggested that villin is regulated by Cdx2. Introduction of Cdx family genes into colorectal SW480 cells revealed that villin is strongly induced strongly by Cdx2, moderately by Cdx1, and marginally by Cdx4. Knockdown of Cdx2 in SW480 cells caused a clear downregulation of villin, and reporter assays showed that HCR-Cdx is crucial for Cdx2-dependent and Brm-dependent villin expression. Immunohistochemical analyses of gastric intestinal metaplasia and cancer revealed that villin and Cdx2 expression are tightly coupled. GST pull-down assays demonstrated a direct interaction between Cdx2 and several SWI/SNF subunits. Chromatin immunoprecipitation analyses showed the recruitment of Cdx2 and Brm around HCR-Cdx. From these results, we concluded that Cdx2 regulates intestinal villin expression through recruiting Brm-type SWI/SNF complex to the villin promoter.
Assuntos
Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Western Blotting , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Sequência Conservada , Genes Reporter , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
We report that a DBHS (Drosophila behaviour, human splicing) family protein, p54(nrb), binds both BRG1 (Brahma-related gene 1) and Brm (Brahma), catalytic subunits of the SWI/SNF (switch/sucrose non-fermentable) chromatin remodelling complex, and also another core subunit of this complex, BAF60a. The N-terminal region of p54(nrb) is sufficient to pull-down other core subunits of the SWI/SNF complex, suggesting that p54(nrb) binds SWI/SNF-like complexes. PSF (polypyrimidine tract-binding protein-associated splicing factor), another DBHS family protein known to directly bind p54(nrb), was also found to associate with the SWI/SNF-like complex. When sh (short hairpin) RNAs targeting Brm were retrovirally expressed in a BRG1-deficient human cell line (NCI-H1299), the resulting clones showed down-regulation of the TERT (telomerase reverse transcriptase) gene and an enhancement of ratios of exon-7-and-8-excluded TERT mRNA that encodes a beta-site-deleted inactive protein. All of these clones display growth arrest within 2 months of the Brm-knockdown. In NCI-H1299 cells, Brm, p54(nrb), PSF and RNA polymerase II phosphorylated on CTD (C-terminal domain) Ser(2) specifically co-localize at a region incorporating an alternative splicing acceptor site of TERT exon 7. These findings suggest that, at the TERT gene locus in human tumour cells containing a functional SWI/SNF complex, Brm, and possibly BRG1, in concert with p54(nrb), would initiate efficient transcription and could be involved in the subsequent splicing of TERT transcripts by accelerating exon-inclusion, which partly contributes to the maintenance of active telomerase.
Assuntos
DNA Helicases/fisiologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição de Octâmero/fisiologia , Splicing de RNA , Proteínas de Ligação a RNA/fisiologia , Telomerase/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMO
RNA-dependent transcriptional silencing (RdTS) has been reported to operate even in human cell lines. It is tempting to speculate that RdTS plays a role in retroviral gene silencing, considering that retroviral RNA transcripts harbor a U3 promoter sequence that is a potentially good source of double-stranded RNAs. To test this possibility, we constructed several model HeLaS3 cell lines expressing GFP driven by murine leukaemia virus (MLV)-long terminal repeat (LTR) and introduced a series of shRNAs that target the U3 region of the MLV-LTR. However, transcriptional gene silencing was not induced in most instances, in spite of the fact that processed shRNA was found in cellular nuclei, indicating that RdTS does not contribute to MLV gene silencing in host cells.
Assuntos
Inativação Gênica , Vírus da Leucemia Murina de Moloney/genética , RNA Interferente Pequeno/fisiologia , Transcrição Gênica , Sequência de Bases , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sequências Repetidas Terminais , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
The transcription factor NF-κB is constitutively activated in many epithelial tumors but few NF-κB inhibitors are suitable for cancer therapy because of its broad biological effects. We previously reported that the d4-family proteins (DPF1, DPF2, DPF3a/b) function as adaptor proteins linking NF-κB with the SWI/SNF complex. Here, using epithelial tumor cell lines, A549 and HeLaS3, we demonstrate that exogenous expression of the highly-conserved N-terminal 84-amino acid region (designated "CT1") of either DPF2 or DPF3a/b has stronger inhibitory effects on anchorage-independent growth than the single knockdown of any d4-family protein. This indicates that CT1 can function as an efficient dominant-negative mutant of the entire d4-family proteins. By in situ proximity ligation assay, CT1 was found to retain full adaptor function, indicating that the C-terminal region of d4-family proteins lacking in CT1 would include essential domains for SWI/SNF-dependent NF-κB activation. Microarray analysis revealed that CT1 suppresses only a portion of the NF-κB target genes, including representative SWI/SNF-dependent genes. Among these genes, IL6 was shown to strongly contribute to anchorage-independent growth. Finally, exogenous CT1 expression efficiently suppressed tumor formation in a mouse xenograft model, suggesting that the d4-family proteins are promising cancer therapy targets.