RESUMEN
Using the MS2 system for labeling mRNA, in this issue, Gallardo et al. (2011) find that telomere lengthening depends on a stable accumulation of multiple telomerase complexes in late S phase and that this process is temporally regulated by Rif1/2 proteins.
RESUMEN
The localization of mRNA appears to facilitate protein sorting, so that proteins are synthesized in specific cellular regions. The spatial information on the mRNA may be transduced by proteins that recognize specific localizing sequences on the 3' end and then chaperone the mRNA, presumably along filaments, to its destination. Additional sequences such as poly(A), or the nascent chains of cytoskeleton-associated proteins, may then anchor mRNAs on the cytoskeleton.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , ARN Mensajero/metabolismo , Animales , Transporte Biológico Activo , Citoplasma/metabolismo , Expresión Génica , Procesamiento Proteico-PostraduccionalRESUMEN
The localization of some mRNAs to distinct intracellular regions is achieved through interactions of the mRNA with cytoskeletal filaments. RNA-cytoskeletal interactions exist that influence the transport, anchoring and translation of mRNA. Recent analysis of RNA movements in living cells suggests the formation of RNA granules and their active transport along microtubules. The anchoring and translation of mRNA may be mediated by interactions with orthogonal networks of F-actin and elongation factor 1alpha.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Citoesqueleto/química , ARN Mensajero/metabolismo , Transporte Biológico/fisiología , Citoesqueleto/metabolismoRESUMEN
RNA localization provides a mechanism for protein targeting within developing or differentiating cells. Specific cis-acting sequences on mRNA mediate this process. Such 'localizer' or 'zipcode' nucleic acid sequences have been restricted to the 3' untranslated region of several mRNAs. The presence of genetic information denoting a spatial component of translation adds a new dimension to gene expression.
Asunto(s)
ARN Mensajero/análisis , AnimalesRESUMEN
The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800-1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hibridación Fluorescente in Situ , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/química , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Transporte de Proteínas , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
RNA-localization mechanisms involve specific sequences in the localized RNA and proteins that bind to these sequences and mediate the interaction with cytoskeletal elements. Until recently, it seemed as though two separate types of mechanisms were operating for mRNA localization--involving interaction with either microtubules or actin microfilaments. However, it is now clear that some of the protein components involved in mRNA localization can participate in both microtubule- and actin-dependent localization pathways. This, combined with new evidence for evolutionary conservation of some of these proteins, suggests a previously unanticipated uniformity in mRNA-localization mechanisms.
Asunto(s)
ARN Mensajero/metabolismo , Citoesqueleto de Actina/fisiología , Actinas/genética , Actinas/fisiología , Animales , Transporte Biológico , Linaje de la Célula , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de Insectos/fisiología , Microtúbulos/fisiología , Proteínas de Unión al ARN/fisiología , Xenopus laevis/anatomía & histologíaRESUMEN
Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.
Asunto(s)
Actinas/genética , Biosíntesis de Proteínas , Erizos de Mar/fisiología , Transcripción Genética , Animales , Peso Molecular , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Erizos de Mar/citología , Especificidad de la EspecieRESUMEN
Actin mRNA is localized in chicken embryo fibroblasts to the distal regions of leading lamellae, but not within the ruffling edges. In this investigation we have addressed the role of actin translation in this process. The translocation of actin mRNA to the cell periphery was studied by monitoring the distribution of actin mRNA in cells during spreading. Within 90 min, actin mRNA moved from a perinuclear to a peripheral distribution. Formation of lamellipodia preceded actin mRNA localization, indicating that localization is not a prerequisite for this event. Neither puromycin (which dissociates ribosomes from mRNA) nor cycloheximide (which stabilizes ribosomes on mRNA) had any effect on this movement of actin mRNA. Anchoring of actin mRNA was studied using cells with peripherally localized actin mRNA. No change in actin mRNA localization was observed for 30 min in the same inhibitors. These data indicate that the presence of the nascent polypeptide is not necessary for translocation of actin mRNA to the cell periphery, or anchoring at that site. This suggests that the mRNA contains information concerning its spatial distribution within the cytoplasm.
Asunto(s)
Actinas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Cicloheximida/farmacología , Sondas de ADN , Emetina/farmacología , Fibroblastos/metabolismo , Cinética , Músculos/metabolismo , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , Puromicina/farmacología , Procesamiento Postranscripcional del ARN , ARN Mensajero/análisis , ARN Mensajero/genética , Fluoruro de Sodio/farmacologíaRESUMEN
We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.
Asunto(s)
Actinas/genética , Membrana Celular/metabolismo , ARN Mensajero/química , Actinas/biosíntesis , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Fenotipo , Seudópodos/ultraestructura , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Tionucleótidos/farmacología , TransfecciónRESUMEN
In chicken embryo fibroblasts (CEFs), beta-actin mRNA localizes near an actin-rich region of cytoplasm specialized for motility, the lamellipodia. This localization is mediated by isoform-specific 3'-untranslated sequences (zipcodes) and can be inhibited by antizipcode oligodeoxynucleotides (ODNs) (Kislauskis, E.H., X.-C. Zhu, and R.H. Singer. 1994. J. Cell Biol. 127: 441-451). This inhibition of beta-actin mRNA localization resulted in the disruption of fibroblast polarity and, presumably, cell motility. To investigate the role of beta-actin mRNA in motility, we correlated time-lapse images of moving CEFs with the distribution of beta-actin mRNA in these cells. CEFs with localized beta-actin mRNA moved significantly further over the same time period than did CEFs with nonlocalized mRNA. Antizipcode ODN treatment reduced this cell translocation while control ODN treatments showed no effect. The temporal relationship of beta-actin mRNA localization to cell translocation was investigated using serum addition to serum-deprived cultures. beta-actin mRNA was not localized in serum-deprived cells but became localized within minutes after serum addition (Latham, V.M., E.H. Kislauskis, R.H. Singer, and A.F. Ross. 1994. J. Cell Biol. 126:1211-1219). Cell translocation increased over the next 90 min, and actin synthesis likewise increased. Puromycin reduced this cell translocation and blocked this induction in cytosolic actin content. The serum induction of cell movement was also inhibited by antizipcode ODNs. These observations support the hypothesis that beta-actin mRNA localization and consequent protein synthesis augment cell motility.
Asunto(s)
Actinas/genética , Movimiento Celular , ARN Mensajero/análisis , Actinas/biosíntesis , Actinas/fisiología , Animales , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Embrión de Pollo , Cicloheximida/farmacología , Citoplasma/química , Citoplasma/ultraestructura , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Oligonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , ARN Mensajero/fisiología , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.
Asunto(s)
Proteínas del Citoesqueleto/genética , Citoesqueleto/metabolismo , ARN Mensajero/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Compartimento Celular , Células Cultivadas , Embrión de Pollo , Inmunohistoquímica , Microscopía Electrónica , Hibridación de Ácido Nucleico , Tubulina (Proteína)/genética , Vimentina/genéticaRESUMEN
Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.
Asunto(s)
Actinas/genética , Actinas/metabolismo , Conos de Crecimiento/metabolismo , Neurotrofina 3/farmacología , ARN Mensajero/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Colchicina/farmacología , Medios de Cultivo , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocalasina D/farmacología , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/enzimología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Actinas/genética , Sustancias de Crecimiento/farmacología , Lisofosfolípidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , ARN Mensajero/metabolismo , Animales , Compartimento Celular/efectos de los fármacos , Embrión de Pollo , Citoesqueleto/metabolismo , Expresión Génica/efectos de los fármacos , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize "individual" poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (< 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a "microdomain" where mRNA is attached and functionally expressed.
Asunto(s)
Actinas/análisis , Poli A/análisis , Poli A/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Animales , Secuencia de Bases , Embrión de Pollo , Citoplasma/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Inmunohistoquímica , Hibridación in Situ , Microscopía Inmunoelectrónica/métodos , Sondas de Oligonucleótidos , Transcripción GenéticaRESUMEN
The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Fibroblastos/ultraestructura , Poli A/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Citoesqueleto de Actina/efectos de los fármacos , Actinas/aislamiento & purificación , Distribución de Chi-Cuadrado , Citocalasina D/farmacología , Citoplasma/ultraestructura , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Histocitoquímica , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Membranas Intracelulares/ultraestructura , Microscopía Fluorescente , Polietilenglicoles/farmacología , Puromicina/farmacología , ARN Mensajero/efectos de los fármacos , Ribonucleasa Pancreática/farmacología , Ribosomas/ultraestructura , Factores de TiempoRESUMEN
By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.
Asunto(s)
Actinina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Uniones Intercelulares/metabolismo , Ligandos , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína de Unión al Tracto de Polipirimidina , Unión Proteica , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Distribución Tisular , Técnicas del Sistema de Dos Híbridos , Vinculina/análogos & derivadosRESUMEN
The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporteros/genética , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plásmidos , Pruebas de Precipitina , Transporte de Proteínas/fisiología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.
Asunto(s)
Distrofia Miotónica/enzimología , Proteínas Quinasas/análisis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , ARN Mensajero/análisis , Adulto , Secuencia de Bases , Células Cultivadas , ADN Complementario , Femenino , Fibroblastos/enzimología , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Músculos/enzimología , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica , Pronóstico , Secuencias Repetitivas de Ácidos Nucleicos , Piel/enzimologíaRESUMEN
We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.
Asunto(s)
Actinas/genética , Compartimento Celular , Citoplasma/metabolismo , Músculos/metabolismo , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Genes Reporteros , Vectores Genéticos , Hibridación in Situ , Datos de Secuencia Molecular , Músculos/citología , Sondas de Oligonucleótidos , ARN Mensajero/genética , Transfección , beta-Galactosidasa/genéticaRESUMEN
The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.