Endogenous Messenger Ribonucleic Acid Research Articles

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

The Expression of Anabolic Cytokines in Intervertebral Discs in Age-Related Degeneration

Quantitative analysis of endogenous messenger ribonucleic acid (mRNA) expression of anabolic cytokines in the anulus fibrosus and nucleus pulposus tissue from the intervertebral discs of young and old rabbits was performed. To measure the expression of anabolic cytokines bone morphogenetic protein-2 (BMP-2), BMP-7, transforming growth factor-beta (TGF-beta), and insulin-like growth factor-I (IGF-I) in the anulus fibrosus and nucleus pulposus tissue from young and old rabbits to determine if there are differences with age. Disc degeneration increases with age and is associated with compromised disc chondrocytic function. Molecules such as BMP-2, BMP-7, TGF-beta, and IGF-I are known to up-regulate disc cell synthesis of key chondrocytic matrix molecules in vitro, and have been proposed as therapeutic agents to prevent disc degeneration. Previous studies have shown that exogenous anabolic cytokines can up-regulate disc-cell function both in vitro and in vivo, however, the endogenous expression of anabolic cytokines in the disc is still unknown. New Zealand white rabbits aged 3 years (old) and 6 months (young) were used. Quantitative real-time polymerase chain reaction was performed to measure the mRNA levels of BMP-2, BMP-7, TGF-beta1, and IGF-I from anulus fibrosus and nucleus pulposus tissue from young and old rabbits. The discs form the young rabbits represent nondegenerated discs, and the discs from the old rabbits represent discs at the onset of degeneration. In the nucleus pulposus, the mRNA levels, given as a ratio of old to young, were 3.6 for BMP-2 (P = 0.004), 61 for BMP-7 (P = 0.02), 4.0 for TGF-beta1 (P = 0.3), and 0.6 for IGF-I (P = 0.2). In the anulus fibrosus, the mRNA levels, given as a ratio of old to young, were 1.6 for BMP-2 (P = 0.07), 4.6 for BMP-7 (P = 0.004), 2.9 for TGF- beta1 (P = 0.01), and 2.0 for IGF-I (P = 0.1). The disc tissue from the old rabbits as compared to the young rabbits showed, in general, significantly higher mRNA levels of endogenous BMP-2, BMP-7, and TGF-beta in both the anulus fibrosus and nucleus pulposus. The similar patterns of up-regulation in gene expression with age shown by these 3 anabolic cytokines suggest a common pathway in terms of regulation and transcription in the early stage of disc degeneration. The knowledge of the age-related pattern in endogenous gene expression of these anabolic cytokines could provide important information for clinical interventional therapy.

Restoration by ribosomal protein S1 of the defective translation in a temperature-sensitive mutant of Escherichia coli K-12: characterization and genetic studies.

A temperature-sensitive mutant of Escherichia coli was isolated that had a temperature-sensitive defect in ribosomal-wash protein(s) required for translation in vitro of E. coli endogenous messenger ribonucleic acid. It was found that 30S ribosomal protein S1 rescued the defect in the ribosomal-wash protein(s) of the mutant and that the complete restoration to the wild-type level was attained when 1 mol of protein S1 was added to 1 mol of 70S ribosome. The mutation, tss, causing such a defect was mapped at 21 min and was closely linked to the pyrD locus, the region of which was entirely different from that of the other genes coding for the many ribosomal proteins of E. coli. These results indicate that the gene specified by this mutation is involved in the function of the 30S ribosomal protein S1.

Protein synthetic activity of membrane-bound and free ribosomes from parathyroid glands of dogs.

1. Membrane-bound and free polyribosomes were isolated from parathyroid glands of normal dogs by a discontinuous density-gradient technique. 2. The conditions necessary for optimum incorporation by bound and free ribosomes of [3-H]-phenylalanine into protein were determined for assays in vitro directed by both endogenous messenger ribonucleic acid (mRNA) and polyuridylic acid [poly(U)]. 3. When the specific cofactors were available in optimum amounts, the rate of incorporation of amino acids into protein was directly proportional to the number of ribosomes present. This applied to assays directed by endogenous mRNA and poly-(U). 4. The results indicate that it is possible to isolate and directly study the protein synthetic activity of membrane-bound and free parathyroid ribosomes.

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.

Some properties of ribosome crystals isolated from hypothermically treated chick embryos.

ABSTRACT Ribosome crystals observed in hypothermically treated chick embryos exist as large dense aggregates and, for this reason, could be purified from a post-nuclear supernatant by centrifugation through a high-density discontinuous sucrose gradient. A ribosome crystal fraction isolated in this way was observed, by electron microscopy, to contain intact crystals, resembling those seen in sectioned whole cells, with only minor contamination from morphologically indistinct particles. The ribonucleic acid (RNA) content of this fraction was found to be the same as ribosomal RNA from mature chickens. However, their resistance to mild RNase treatment, lack of transfer RNA and endogenous messenger RNA activity indicate that such ribosome crystals are not a specific type of polysome. The intertetramer bonds (i.e. bonds between tetramers in a crystal array) are much more susceptible to mild protease treatment than to treatment with ribonuclease (RNase), indicating a dependence on protein-protein interactions for their formation.

Translation of synthetic and endogenous messenger ribonucleic acid in vitro by ribosomes and polyribosomes from Clostridium pasteurianum.

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.

Synthesis of vaccinia viral proteins in cytoplasmic extracts. I. Incorporation of radioactively labeled amino acids into polypeptides.

Polypeptide synthesis has been studied in cell-free systems prepared from vaccinia virus-infected and uninfected HeLa cells. Cytoplasmic extracts containing endogenous messenger ribonucleic acid were used. Amino acid incorporation into hot trichloroacetic acid-precipitable material was linear for 15 to 20 min at 37 C. The initial rate of protein synthesis was approximately 15% of the rate in intact cells. Optimal conditions for polypeptide synthesis were similar in cell-free systems prepared from infected or uninfected cells. Requirements for an energy source and Mg(++) were demonstrated. The optimal Mg(++) concentration was 4 to 5 mm. Ribonuclease, puromycin, and cycloheximide were inhibitory. The molecular weights of the polypeptides labeled in the cell-free systems, as determined by gel filtration in 5 m guanidine hydrochloride, ranged from 16,000 to above 68,000. Polyacrylamide gel electrophoresis indicated that the polypeptides labeled in cell-free extracts of uninfected and infected cells were different. The latter closely corresponded in electrophoretic mobility with the viral polypeptides made in intact, infected cells.

Streptomycin action: greater inhibition of Escherichia coli ribosome function with exogenous than with endogenous messenger ribonucleic acid.

Inhibition of protein synthesis by streptomycin was tested in extracts from a strain of Escherichia coli sensitive to streptomycin. Three kinds of messenger ribonucleic acid (RNA) were employed: endogenous cellular RNA, extracted cellular RNA, and phage R17 RNA. Protein synthesis directed by extracted cellular RNA was inhibited three- to fourfold more than protein synthesis directed by endogenous RNA. With R17 RNA as messenger, nearly total inhibition of protein synthesis at initiation was again observed. The greater inhibition of function of extracted RNA, which must initiate new polypeptide chains in vitro, is in accord with the observation that in whole cells streptomycin blocks ribosomes at an early stage in protein synthesis. When streptomycin was added at successively later times during protein synthesis, the subsequent inhibition was progressively less. This was observed with either extracted cellular RNA or phage R17 RNA. A model is presented that can explain the less drastic inhibition by streptomycin of messenger RNA that is already functioning on ribosomes.

Protein synthesis in a cell-free extract from Staphylococcus aureus.

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 x g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.

Studies on the Removal of Endogenous Messenger Ribonucleic Acid Activity from Rat Liver Microsomes: EFFECT OF REMOVAL ON POLYURIDYLIC ACID-DIRECTED INCORPORATION OF PHENYLALANINE

Abstract Rat liver microsomes preincubated for 12 min with all the factors necessary for amino acid incorporation do not incorporate phenylalanine in the absence of polyuridylic acid. In the presence of polyuridylic acid, preincubated microsomes show a 3- to 4-fold greater polyuridylic acid-directed phenylalanine incorporation than nonpreincubated microsomes. By varying preincubation time, temperature, and magnesium ion, adenosine triphosphate, or guanosine triphosphate concentration, the amount of phenylalanine incorporated during preincubation and the phenylalanine-incorporating activity remaining after preincubation are altered. Under these various conditions the response of preincubated microsomes to polyuridylic acid is shown to be directly related to the amount of amino acid incorporated during preincubation and inversely related to the amino acid-incorporating activity remaining after preincubation. Preincubated microsomes are more sensitive to low levels of polyuridylic acid and require higher levels of polyuridylic acid to saturate their capacity to incorporate phenylalanine. Furthermore, preincubated microsomes from which endogenous messenger ribonucleic acid activity has been removed bind greater amounts of polyuridylic acid.