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Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.  相似文献   
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The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.  相似文献   
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Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with 32Pi. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of 32P) and not sedimentable (supernatant) material. About 90% of 32P in the supernatant fraction was sedimentable at 100,000 g (disperse chromatin). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of 32P. The 32P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the 32P-labeled nuclear proteins into 12 major bands in the Mr range of 12,000–120,000. The incorporation of 32P into most bands reached a steady-state within 5–10 min of incubation with 32Pi and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an Mr 95,000 phosphoprotein. Two of the fastest moving 32P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving 32P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.  相似文献   
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87Rb, 23Na and 31P nuclear magnetic resonance (NMR) were used to monitor changes in renal cations and energetics during the induction of hypoxia in the isolated perfused rat kidney. The NMR-determined unidirectional Rb+ flux in normoxic kidneys was shown to be a good measure of net intracellular K+ influx in the perfused rat kidney model. The changes in 87Rb, 23Na and 31P spectra following the induction of hypoxia are consistent with hypoxic depletion of intracellular adenosine triphosphate (ATP) and a subsequent decrease in Na-K-ATPase transport activity. The exponential rate constant for 87Rb+ efflux measured during Rb+ uptake in normoxic kidneys (0.12 +/- 0.01 min-1) was not significantly different to the rate constant for 87Rb+ efflux during the induction of hypoxia (0.16 +/- 0.07 min-1). We conclude that there is no direct effect of hypoxia on renal cellular membrane integrity and that renal cell sensitivity to hypoxia is due to an inability to sustain cellular ion gradients following depletion of intracellular ATP.  相似文献   
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A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold.  相似文献   
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Renal reabsorption of urea was studied in 8 9-month old reindeer calves fed low protein (19–34 g crude protein, mostly lichens) and high protein (68–69 g crude protein, lichens + soybean meal) diets. Low protein diets fed for a 3-month period resulted in an average renal reabsorption of 93% of the filtered urea, while only 50 % was reabsorbed on the high protein ration. It was calculated that if the reabsorbed urea was used completely for protein synthesis in the rumen, 4 g crude protein could be made daily in the lichen fed animals. This amount would be a very significant contribution to the nitrogen economy of animals which are usually in a negative nitrogen balance when lichens are the main food consumed.  相似文献   
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