Association of mRNA with the cytoskeletal framework: its role in the regulation of gene expression.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J S Pachter
Publications and source records attributed to J S Pachter.
"In situ" translation: use of the cytoskeletal framework to direct cell-free protein synthesis.
We have developed a novel, "in situ" translation system derived from cultured cells that are subject to mild detergent extraction. By using a low concentration of nonionic detergent to gently permeabilize cells while they remain adherent to a substrate, cytoskeletal frameworks are obtained that are devoid of membraneous barriers yet retain much the same topological arrangement of mRNA, ribosomes and cytostructure that exists "in vivo". Data indicate that when these cytoskeletal frameworks are supported by a ribosome-depleted, nuclease-treated, reticulocyte lysate supernatant, they are capable of resuming translation of their attached polysomes for at least 40 minutes. Emulsion autoradiography of ongoing protein synthesis demonstrates that protein synthetic activity is ubiquitous throughout the population of extracted cells, and not confined to a less well-extracted subset. Computer-assisted, two-dimensional gel analysis reveals that the pattern of proteins produced by such extracted cells is approximately 70% coincident with that produced by unextracted cells, including proteins of molecular weight as great as 200 kilodaltons. Furthermore, a continued increase in intensity of almost all proteins during the first 40 minutes of translation suggests that translational re-initiation, in addition to polysome run-off, is also taking place. Collectively, these findings indicate that much of the translational machinery remains both intact and competent in this cytoskeletal-based translation system. As such, this system should prove extremely useful in identifying molecular factors operant during certain types of translation control and in further examining the role played by the cytoskeleton in regulating gene expression.
Animals↗
Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides.
The expression of tubulin polypeptides in animal cells is controlled by an autoregulatory mechanism whereby increases in the tubulin subunit concentration result in rapid and specific degradation of tubulin mRNAs. We have now determined that the sequences that are necessary and sufficient to specify mouse beta-tubulin mRNAs as substrates for this autoregulated instability reside within the first 13 translated nucleotides (which encode the first four beta-tubulin amino acids Met-Arg-Glu-Ile). This domain has been functionally conserved throughout evolution, inasmuch as sequences isolated from the analogous region of human, chicken, and yeast beta-tubulin mRNAs also confer autoregulation. Further, for an RNA to be a substrate for regulation, not only must it carry the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to codon 41.
Amino Acid Sequence↗
Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs.
We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.
Animals↗
Intracellular distribution of mammalian stress proteins. Effects of cytoskeletal-specific agents.
Following a brief period of heat stress, the two highly conserved mammalian stress proteins, hsp68 and 70, were examined with respect to their intracellular locations. In four independent cell lines, hsp68 and 70 were found to partition into both Triton X-100-soluble and insoluble fractions as assessed by two-dimensional gel analysis of newly synthesized polypeptides, whereas a fifth cell line showed these proteins only in the Triton X-100-insoluble fraction. In addition, a previously described cell fractionation technique was utilized to gain information regarding the segregation of the two major mammalian stress proteins, hsp68 and 70, into distinct biochemically and morphologically characterized subcellular compartments of PtK2-epithelial cells. Two cytoskeletal-specific agents, taxol and colchicine, were also probed for their effects on the disposition of these polypeptides. Under our conditions of acute heat exposure, hsp68, 70 and their isoforms were globally distributed in all subcellular fractions examined, with a few notable exceptions in drug-treated cells. Colchicine, a microtubule-depolymerizing drug, inhibited the association of hsp68 and its variants with the double-detergent-extractable labile "cytoskeleton," whereas taxol, a microtubule-stabilizing agent, in some manner, facilitated the transit of hsp68 and its isovariants from a cytoplasmic to nuclear domain. Degree of cell density is a factor which influences the synthesis of various cytoskeletal proteins; therefore, we studied the effect of cell confluency on the disposition of mammalian stress proteins hsp68 and 70 in human FS-4 fibroblasts. In confluent cultures, where cell-cell contact was maximal, we observed the appearance of a previously undetected polypeptide which was not found in sparsely populated cultures. This protein may represent a post-translationally modified isoform of a preexisting heat shock protein, or perhaps, a novel stress protein.
Alkaloids↗
alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.
In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.
Animals↗
beta-Internexin, a ubiquitous intermediate filament-associated protein.
In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).
Animals↗
Associated proteins as possible cross-linkers in the neuronal cytoskeleton.
Explore the source record for details and available documents.
Animals↗
The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve.
The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.
Aging↗
A batchwise purification procedure of neurofilament proteins.
A rapid batchwise purification procedure for neurofilament proteins from bovine spinal cord is described. A crude filament fraction can be obtained by treating the tissue with Triton X-100, followed by centrifugation through sucrose. From this crude filament fraction the protein is processed through a batch purification procedure using hydroxyapatite in 8 M urea. With this procedure, approximately 0.5 g of purified neurofilament protein is obtained from a single bovine spinal cord in less than 3 days.
Animals↗
Brain tryptophan in isolated aggressive mice.
Circadian variations in whole brain tryptophan level and food intake were investigated in both normally grouped and isolated male Albino-Swiss mice, maintained on a 12:12 light-dark regimen (lights on 08.00--20.00). A significant diurnal rhythm in brain tryptophan level was evidenced only by grouped animals, in contrast to the relatively sustained and significantly lower tryptophan level found to exist in isolated mice. Significant, daily rhythms in food intake, for both isolated and grouped mice were found to exist, that were highly correlated in phase. Isolated mice, however, consistently demonstrated an increased food consumption throughout the 24-hour period. These results suggest that a possible alteration in tryptophan metabolism exists in isolated mice, as tryptophan, an essential amino acid, is obtained by mammals only through their diet.
Aggression↗