RESUMEN
Skeletal muscle hypertrophy and regeneration are important adaptive responses to both physical activity and pathological stimuli. Failure to maintain these processes underlies the loss of skeletal muscle mass and strength that occurs with ageing and in myopathies. Here we show that stable expression of a gene encoding insulin-like growth factor 1 (IGF-1) in C2C12 skeletal muscle cells, or treatment of these cells with recombinant IGF-1 or with insulin and dexamethasone, results in hypertrophy of differentiated myotubes and a switch to glycolytic metabolism. Treatment with IGF-1 or insulin and dexamethasone mobilizes intracellular calcium, activates the Ca2+/calmodulin-dependent phosphatase calcineurin, and induces the nuclear translocation of the transcription factor NF-ATc1. Hypertrophy is suppressed by the calcineurin inhibitors cyclosporin A or FK506, but not by inhibitors of the MAP-kinase or phosphatidylinositol-3-OH kinase pathways. Injecting rat latissimus dorsi muscle with a plasmid encoding IGF-1 also activates calcineurin, mobilizes satellite cells and causes a switch to glycolytic metabolism. We propose that growth-factor-induced skeletal-muscle hypertrophy and changes in myofibre phenotype are mediated by calcium mobilization and are critically regulated by the calcineurin/NF-ATc1 signalling pathway.
Asunto(s)
Calcineurina/metabolismo , Calcio/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Proteínas Nucleares , Transducción de Señal , Células 3T3 , Animales , Cardiomegalia/metabolismo , Diferenciación Celular , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Glicoproteínas/farmacología , Hipertrofia , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Ratones , Músculo Esquelético/efectos de los fármacos , Factores de Transcripción NFATC , Neurregulinas , Monoéster Fosfórico Hidrolasas/metabolismo , Plásmidos , Ratas , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop). Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.
Asunto(s)
Desoxirribonucleótidos/química , Precursores del ARN/metabolismo , Empalme del ARN/fisiología , ARN Nuclear Pequeño/química , Secuencia de Bases , Desoxirribonucleótidos/síntesis química , Radical Hidroxilo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Nuclear Pequeño/metabolismo , Empalmosomas/química , Levaduras/metabolismoRESUMEN
Specific yeast tRNA(Phe) hydrolysis brought about by europium ions has been studied in detail using the 32P-end-labeled tRNA and polyacrylamide gel electrophoresis. The dependence of the induced cleavages on pH, temperature and concentration of the europium ions has been determined. Europium hydrolyzes yeast tRNA(Phe) in the D-loop at phosphates 16 and 18, and the anticodon loop of phosphates 34 and 36. The two D-loop cuts are thought to take place from two distinct europium binding sites, while the two anticodon loop cleavages from a single site. Eight other members of the lanthanide series and ytrium give basically the same pattern of cleavages as europium. The specific cleavages taking place in the anticodon loop occur in an intramolecular mode from the lanthanide binding site that has not been found in yeast tRNA(Phe) crystal structure. It appears from the comparison of the europium-promoted cuts with those generated by magnesium and lead that the former two ions give more similar but not identical cleavage patterns. The usefulness of the specific cleavages induced by lanthanides for probing their own and magnesium binding sites in tRNA is discussed.
Asunto(s)
Metales de Tierras Raras/análisis , ARN de Transferencia Aminoácido-Específico/análisis , ARN de Transferencia de Fenilalanina/análisis , Saccharomyces cerevisiae/análisis , Sitios de Unión/efectos de los fármacos , Europio/análisis , Concentración de Iones de Hidrógeno , Hidrólisis , Plomo/análisis , Magnesio/análisis , TemperaturaRESUMEN
Yeast tRNA(Phe) and tRNA(Phe-Y) are cleaved by single strand-specific endonuclease S1 at the same positions within the anticodon loop (phosphates 34, 36 and 37) and at the 3'-terminus (phosphates 75 and 76). The efficiency of the anticodon loop hydrolysis is much higher in tRNA(Phe-Y) while the cutting at the 3'-terminus is not influenced considerably by the Y-base1 removal from yeast tRNA(Phe). The effect of the Y-base excision on the structure of the anticodon loop is discussed on the basis of the S1 digestion studies as well as other relevant results.
Asunto(s)
Aspergillus oryzae/genética , Aspergillus/genética , Conformación de Ácido Nucleico , Sondas ARN , ARN de Transferencia Aminoácido-Específico , ARN de Transferencia de Fenilalanina , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Anticodón , Aspergillus oryzae/enzimología , HidrólisisRESUMEN
The specificity of magnesium ion-induced hydrolysis of yeast tRNAPhe in solution was studied as a function of the excess of Mg(II) ions and pH. The major cuts at phosphates 16 and 20 as well as minor cleavages at phosphates 17, 18, 21, 34 and 36 occur at all pH values in the range of 8.0-9.5, and at a molar excess of magnesium ions over the tRNA ranging from 125 to 5000. In yeast tRNA(Phe)-Y the efficiency of the anticodon and D-loop cleavages is considerably decreased while the differently modified Y-base of yellow lupin tRNA(Phe) lowers the specificity of the weak anticodon loop cleavages. The mechanism of the Mg(II)-induced cleavages is discussed on the basis of yeast tRNA(Phe) crystal structure data, and the two major D-loop cleavages are thought to be effected from two distinct magnesium binding sites. The possibility of probing the environments of magnesium binding sites in tRNAs by the induced cleavages is demonstrated, and the relevance of magnesium-induced tRNA cleavages to RNA catalysis is discussed.
Asunto(s)
Magnesio/farmacología , ARN de Transferencia de Fenilalanina/efectos de los fármacos , Levaduras/genética , Fabaceae/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Conformación de Ácido Nucleico , Plantas Medicinales , ARN de Transferencia de Fenilalanina/química , Soluciones , Especificidad de la EspecieRESUMEN
The patterns of limited hydrolysis of yeast tRNA(Phe) and tRNA(-YPhe) by double strand-specific ribonuclease V1 show some differences in cleavage of both the acceptor stem and the anticodon stem. These regions are considerably better substrates for RNase V1 in tRNA(-YPhe) than in tRNA(Phe). The results are interpreted in favour of conformational changes taking place in yeast tRNA(Phe) upon the Y-base1 removal.
Asunto(s)
Endorribonucleasas , Conformación de Ácido Nucleico , ARN de Transferencia Aminoácido-Específico , ARN de Transferencia de Fenilalanina , Saccharomyces cerevisiae/genética , Animales , Anticodón , Bovinos , HidrólisisRESUMEN
The Pb, Eu and Mg-induced cleavages in E. coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe. The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E. coli tRNAPhe hydrolysis. The latter tRNA is cleaved with much lower yield in the D-loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs. The possible location of tight Pb, Eu and Mg binding sites in E. coli tRNAPhe is discussed on the basis of the specific hydrolysis data.
Asunto(s)
Metales/metabolismo , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Escherichia coli/metabolismo , Europio/metabolismo , Fabaceae/metabolismo , Hidrólisis , Plomo/metabolismo , Magnesio/metabolismo , Metales/farmacología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plantas Medicinales , Saccharomyces cerevisiae/metabolismoRESUMEN
The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.