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1.
Condeelis J Singer RH 《Biology of the cell / under the auspices of the European Cell Biology Organization》2005,97(1):97-110
beta-Actin mRNA is localized near the leading edge in several cell types where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3'UTR (untranslated region), the 'zipcode'. Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zipcode) in the 3'UTR leads to delocalization of beta-actin mRNA, alteration of cell phenotype and a decrease in cell motility. The dynamic image analysis system (DIAS) used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zipcode showed that net pathlength and average speed of antisense-treated cells were significantly lower than in sense-treated cells. This suggests that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zipcode-directed antisense oligonucleotides. We postulate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement. Hence the physiological consequences of beta-actin mRNA delocalization affect the stability of the cell phenotype. 相似文献
2.
Two ZBP1 KH domains facilitate beta-actin mRNA localization,granule formation,and cytoskeletal attachment 总被引:1,自引:0,他引:1
Farina KL Huttelmaier S Musunuru K Darnell R Singer RH 《The Journal of cell biology》2003,160(1):77-87
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility. 相似文献
3.
Wei Gu Feng Pan Honglai Zhang Gary J Bassell Robert H Singer 《The Journal of cell biology》2002,156(1):41-51
The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3' untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of beta-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA. 相似文献
4.
A F Ross Y Oleynikov E H Kislauskis K L Taneja R H Singer 《Molecular and cellular biology》1997,17(4):2158-2165
Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA. 相似文献
5.
Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility 总被引:15,自引:0,他引:15
Zhang HL Eom T Oleynikov Y Shenoy SM Liebelt DA Dictenberg JB Singer RH Bassell GJ 《Neuron》2001,31(2):261-275
Neurotrophin regulation of actin-dependent changes in growth cone motility may depend on the signaling of beta-actin mRNA transport. Formation of an RNP complex between the beta-actin mRNA zipcode sequence and Zipcode Binding Protein 1 (ZBP1) was required for its localization to growth cones. Antisense oligonucleotides to the zipcode inhibited formation of this RNP complex in vitro and the neurotrophin-induced localization of beta-actin mRNA and ZBP1 granules. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bidirectional movements of granules in neurites that were inhibited by antisense treatment, as visualized by FRAP analysis. NT-3 stimulation of beta-actin protein localization was dependent on the 3'UTR and inhibited by antisense treatment. Growth cones exhibited impaired motility in the presense of antisense. These results suggest a novel mechanism to influence growth cone dynamics involving the regulated transport of mRNA. 相似文献
6.
Beta-actin mRNA localization is regulated by signal transduction mechanisms 总被引:8,自引:2,他引:6
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《The Journal of cell biology》1994,126(5):1211-1219
Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407- 415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility. 相似文献
7.
BACKGROUND: The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, beta-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. RESULTS: We demonstrate that a signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing beta-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, we found that inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibited beta-actin mRNA from localizing in response to growth factors. CONCLUSIONS: We therefore conclude that the sorting of beta-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and postulate that polarized actin bundles direct the mRNA to the leading edge of the cell. 相似文献
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The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions. 相似文献
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We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells. 相似文献
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Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. 总被引:118,自引:14,他引:104
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We report the complete nucleotide sequence of a human beta actin cDNA. Both the 5' and 3' untranslated regions of the sequence are similar (greater than 80%) to the analogous regions of the rat beta-actin gene reported by Nudel et al (1983). When a segment of the 3' untranslated region is used as a radiolabelled probe, strong hybridization to chick beta actin mRNA is seen. This conservation of sequences suggests that strong selective pressures operate on non-translated segments of beta actin mRNA. 相似文献
15.
Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes 总被引:4,自引:0,他引:4
We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations. 相似文献
16.
Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5'' end of the chicken alpha-skeletal actin gene. 总被引:35,自引:4,他引:31
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alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression. 相似文献
17.
Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation. 相似文献
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Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes 总被引:8,自引:0,他引:8
The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization. 相似文献
20.
Faouzi Baklouti Shu-Ching Huang Tom J. Vulliamy Jean Delaunay Edward J. Benz Jr. 《Genomics》1997,39(3):289
Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3′-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3′ UTR, over 3 kb, accounts for one-half of the mature mRNA. 相似文献