Accumulation Of Ribosomal RNA Research Articles

Glucose ingestion before and after resistance training sessions does not augment ribosome biogenesis in healthy moderately trained young adults

PurposeResistance training-induced skeletal muscle hypertrophy seems to depend on ribosome biogenesis and content. High glucose treatment may augment ribosome biogenesis through potentiating resistance training-induced adaptations. This was investigated with total RNA and ribosomal RNA abundances as main outcomes, with relevant transcriptional/translational regulators (c-Myc/UBF/rpS6) as a secondary outcome.MethodsSixteen healthy, moderately trained individuals [male/female, n = 9/7; age, 24.1 (3.3)] participated in a within-participant crossover trial with unilateral resistance training (leg press and knee extension, 3 sets of 10 repetitions maximum) and pre- and post-exercise ingestion of either glucose (3 × 30 g, 90 g total) or placebo supplements (Stevia rebaudiana, 3 × 0.3 g, 0.9 g total), together with protein (2 × 25 g, 50 g total), on alternating days for 12 days. Six morning resistance exercise sessions were conducted per condition, and the sessions were performed in an otherwise fasted state. Micro-biopsies were sampled from m. vastus lateralis before and after the intervention.ResultsGlucose ingestion did not have beneficial effects on resistance training-induced increases of ribosomal content (mean difference 7.6% [− 7.2, 24.9], p = 0.34; ribosomal RNA, 47S/18S/28S/5.8S/5S, range 7.6–37.9%, p = 0.40–0.98) or levels of relevant transcriptional or translational regulators (c-MYK/UBF/rpS6, p = 0.094–0.292). Of note, both baseline and trained state data of total RNA showed a linear relationship with UBF; a ∼14% increase in total RNA corresponded to 1 SD unit increase in UBF (p = 0.003).ConclusionGlucose ingestion before and after resistance training sessions did not augment ribosomal RNA accumulation during twelve days of heavy-load resistance training in moderately trained young adults.

Nucleolar localization of c‐Jun

Nucleoli are well defined for their function in ribosome biogenesis, but only a small fraction of the nucleolar proteome has been characterized. Here, we report that the proto-oncogene, c-Jun, is targeted to the nucleolus. Using live cell imaging in myogenic cells, we document that the c-Jun basic domain contains a unique, evolutionarily conserved motif that determines nucleolar targeting. Fos family Jun dimer partners, such as Fra2, while nuclear, do not co-localize with c-Jun in the nucleolus. A point mutation in c-Jun that mimics Fra2 (M260E) in its Nucleolar Localization sequence (NoLS) results in loss of c-Jun nucleolar targeting while still preserving nuclear localization. Fra2 can sequester c-Jun in the nucleoplasm, indicating that the stoichiometric ratio of heterodimeric partners regulates c-Jun nucleolar targeting. Finally, nucleolar localization of c-Jun modulates nucleolar architecture and ribosomal RNA accumulation. These studies highlight a novel role for Jun family proteins in the nucleolus, having potential implications for a diverse array of AP-1-regulated cellular processes.

AS1411-conjugated gold nanoparticles affect cell proliferation through a mechanism that seems independent of nucleolin

G-rich oligonucleotide, AS1411, has been shown to interact with nucleolin and to inhibit cancer cell proliferation and tumor growth. This antiproliferative action is increased when AS1411 is conjugated to different types of nanoparticles. However, the molecular mechanisms are not known. In this work, we show in several cell lines that optimized AS1411-conjugated gold nanoparticles (GNS-AS1411) inhibit nucleolin expression at the RNA and protein levels. We observed an alteration of the nucleolar structure with a decrease of ribosomal RNA accumulation comparable to what is observed upon nucleolin knock down. However, the expression of genes involved in cell cycle and the cell cycle blockage by GNS-AS1411 are not regulated in the same way as that in cells where nucleolin has been knocked down. These data suggest that the anti-proliferative activity of GNS-AS1411 is not the only consequence of nucleolin targeting and down-regulation.

Role of ribosome assembly in Escherichia coli ribosomal RNA degradation.

DEAD-Box proteins (DBPs) constitute a prominent class of RNA remodeling factors that play a role in virtually all aspects of RNA metabolism. To better define their cellular functions, deletions in the genes encoding each of the Escherichia coli DBPs were combined with mutations in genes encoding different Ribonucleases (RNases). Significantly, double-deletion strains lacking Ribonuclease R (RNase R) and either the DeaD or SrmB DBP were found to display growth defects and an enhanced accumulation of ribosomal RNA (rRNA) fragments. As RNase R is known to play a key role in removing rRNA degradation products, these observations initially suggested that these two DBPs could be directly involved in the same process. However, additional investigations indicated that DeaD and SrmB-dependent rRNA breakdown is caused by delays in ribosome assembly that increase the exposure of nascent RNAs to endonucleolytic cleavage. Consistent with this notion, mutations in factors known to be important for ribosome assembly also resulted in enhanced rRNA breakdown. Additionally, significant levels of rRNA breakdown products could be visualized in growing cells even in the absence of assembly defects. These findings reveal a hitherto unappreciated mechanism of rRNA degradation under conditions of both normal and abnormal ribosome assembly.

Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

The role of skeletal muscle mTOR in the regulation of mechanical load-induced growth.

Chronic mechanical loading (CML) of skeletal muscle induces compensatory growth and the drug rapamycin has been reported to block this effect. Since rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), many have concluded that mTOR plays a key role in CML-induced growth regulatory events. However, rapamycin can exert mTOR-independent actions and systemic administration of rapamycin will inhibit mTOR signalling in all cells throughout the body. Thus, it is not clear if the growth inhibitory effects of rapamycin are actually due to the inhibition of mTOR signalling, and more specifically, the inhibition of mTOR signalling in skeletal muscle cells. To address this issue, transgenic mice with muscle specific expression of various rapamycin-resistant mutants of mTOR were employed. These mice enabled us to demonstrate that mTOR, within skeletal muscle cells, is the rapamycin-sensitive element that confers CML-induced hypertrophy, and mTOR kinase activity is necessary for this event. Surprisingly, CML also induced hyperplasia, but this occurred through a rapamycin-insensitive mechanism. Furthermore, CML was found to induce an increase in FoxO1 expression and PKB phosphorylation through a mechanism that was at least partially regulated by an mTOR kinase-dependent mechanism. Finally, CML stimulated ribosomal RNA accumulation and rapamycin partially inhibited this effect; however, the effect of rapamycin was exerted through a mechanism that was independent of mTOR in skeletal muscle cells. Overall, these results demonstrate that CML activates several growth regulatory events, but only a few (e.g. hypertrophy) are fully dependent on mTOR signalling within the skeletal muscle cells.

RIBOSOME SYNTHESIS IN TETRAHYMENA: A QUANTITATIVE ANALYSIS

We have performed a detailed quantitative analysis of the transcription and accumulation of ribosomal RNA and ribosomal protein mRNA in the ciliated protozoan Tetrahymena thermophila during changes in growth conditions, and found that: (1) nutritional downshifts lead to a rapid decrease in transcriptional activity whereas nutritional upshifts lead to rapid restoration of transcriptional activity, (2) starvation leads to decreased translation of ribosomal protein mRNA and (3) the rate of ribosomal protein mRNA degradation decreases after a nutritional upshift. We present evidence that the proximal promoters of two ribosomal protein genes and the ribosomal RNA gene compete for binding of nuclear factor(s) in vitro, suggesting that the coordinated regulation of these genes may involve a common set of transcriptional regulators.

A temperature-sensitive mutant ofEscherichia coliaffected in the alpha subunit of RNA polymerase

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min. on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44 degrees C. The thermostability at 44 degrees C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37 degrees C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed.

Selective inhibition of 28S ribosomal RNA in macrophages activated by interferon-gamma or -beta.

We have investigated the metabolism of RNA in mouse peritoneal exudate macrophages activated by interferon (IFN)-gamma or -beta. Both species of IFN induce cytotoxic activity in macrophages. We observed a decrease in the incorporation of [3H]-uridine into total RNA in macrophages treated with doses of IFN that induce cytotoxic activity. IFN-gamma was 100-fold to 1000-fold more potent that IFN-beta in inhibiting RNA synthesis. [3H]Uridine-labeled RNA was purified from IFN-activated and control macrophages and was size fractionated on agarose gels. Macrophages activated by either IFN-gamma or IFN-beta had an imbalanced accumulation of 28S ribosomal RNA compared with their accumulation of 18S ribosomal RNA. Pulse-chase experiments suggested that IFN induced a selective inhibition of the processing of 28S ribosomal RNA. These results provide the first evidence that IFN can modulate ribosomal gene expression at the post-transcriptional level. Moreover, they indicate that inhibition of 28S ribosomal RNA accumulation in macrophages is a molecular event triggered by IFN-gamma, as well as IFN-beta.

Imbalanced accumulation of ribosomal RNA in macrophages activated in vivo or in vitro to a cytolytic stage.

Previous studies have shown that peritoneal murine macrophages activated in vivo and in vitro to a tumoricidal stage have a depressed rate of RNA synthesis. In attempting to clarify the differences in RNA metabolism between noncytotoxic and tumoricidal macrophages, we have studied the relative accumulation of various species of RNA in macrophages activated in vivo and in vitro with the use of agarose gel electrophoresis. Macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines (LK) and traces of lipopolysaccharide (LPS) have an imbalanced accumulation of mature ribosomal RNA (rRNA), with a decreased accumulation of 28S rRNA compared to 18S rRNA. In contrast, macrophages primed in vitro with LK free of detectable endotoxins that exhibit suppressive rather than tumoricidal activity do not manifest a decreased 28S:18S rRNA ratio. The conclusion that the decreased 28S:18S rRNA ratio was associated with the activation of macrophages to a cytolytic stage was supported by the finding that cytotoxic macrophages activated in vivo by i.p. injection of Propionibacterium acnes (formerly designated C. parvum) also demonstrated a decreased accumulation of 28S comparable with that observed in in vitro-activated macrophages. Moreover, activated macrophages that lost their cytolytic activity upon prolonged in vitro culture had an augmented accumulation of 28S rRNA. These results provide the first direct evidence that the expression of cytolytic activity is associated with modulation of a specific class of RNA. The unbalanced accumulation of rRNA appears to be a late molecular event in the activation process occurring during the transition from primed to cytotoxic macrophages, because inflammatory and primed macrophages had normal rRNA accumulation. A model of macrophage activation accounting for these results is proposed.

The action of exogenous abscisic and gibberellic acids on gene expression in germinating castor beans

Exogenously applied abscisic acid inhibits isocitrate-lyase activity of the endosperm during germination of castor-bean seeds. Amounts of isocitrate-lyase mRNA have been estimated by immunoprecipitation of in-vitro-translated polypeptide products. Exogenous abscisic acid leads to an inhibition of isocitrate lyase-mRNA accumulation. A large proportion of this effect of the growth factor may be accounted for by its action in inhibiting the overall accumulation of ribosomal RNA and total mRNA. However, the effect of abscisic acid on protein synthesis is not general, as the production of some mRNAs was stimulated. The major mRNA stored in the dry seed, coding for a 25600-Mr polypeptide that normally disappears within the first 12 h of germination, exhibited high levels in abscisic-acid-treated endosperms throughout the germination period. Three complementary DNA clones, of which two clones are complementary to isocitrate lyase, have been used to measure levels of transcripts during seed germination. The accumulation of both transcripts was inhibited by exogenous abscisic acid. The data strongly indicate that the action of abscisic acid on isocitrate lyase synthesis is either to inhibit the transcription, or to increase the transcript turnover. Exogenous gibberellic acid is able to counteract the inhibitory effects of abscisic acid.

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.

Only one of the two signals required for initiation of the cell cycle is associated with cellular accumulation of ribosomal RNA

Two intracellular functions are elicited by the serum component in culture media in order to initiate the cell cycle: “competence” and “progression”. Although both functions have to be present simultaneously for start of cell division, it is shown here for Swiss 3T3 cells that only one of them, the progression signal, is associated with reaccumulation of ribosomal RNA. This result points to the cellular complement of ribosomes as one of the limiting parameters for cell division.

Unbalanced ribosomal protein synthesis in a strain of Escherichia coli containing a cloned, truncated 16-S ribosomal RNA gene.

An Escherichia coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis. Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone. The rate of total protein synthesis, however, appears to be normal. In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated. The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7). Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S12 was moderately over-produced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.

Induction of casein synthesis by prolactin and inhibition by progesterone in the pseudopregnant rabbit treated by colchicine without any simultaneous variations of casein mRNA concentration.

Prolactin injected into pseudopregnant rabbits induced casein synthesis and it provoked an accumulation of casein mRNA and of ribosomal RNA. Colchicine, which has been shown to block the prolactin signal, totally prevented the accumulation of beta-casein mRNA, when injected with the hormone. However, the drug did not hamper the initiation of casein synthesis and the accumulation of ribosomal RNA. The effect of prolactin injected with colchicine on casein synthesis was totally abrogated by progesterone administered simultaneously and it was essentially unmodified by glucocorticoids. These results suggest that prolactin controls casein gene expression by supporting the accumulation of casein mRNAs and by stimulating the translation of these mRNAs, through independent mechanisms. Progesterone which is known to prevent the accumulation of casein mRNAs is also a potent inhibitor of casein mRNA translation whereas glucocorticoids exert their effect essentially by favouring the accumulation of casein mRNA but not by modifying their translation efficiency.

Transcriptional regulation of ribosomal RNA accumulation during L 6E 9 myoblast differentiation

The rat myoblast cell line L 6E 9 under appropriate culture conditions is a uniform population of cycling cells which can be induced to differentiate into pure populations of myotubes. These myotubes have irreversibly withdrawn from the cell cycle but continue to synthesize messenger RNA and protein at the pre-fusion level. We have used this cell line to study the regulation of ribosome synthesis during differentiation of the growing myoblasts into non-growing myotubes. We have found that ribosome accumulation is reduced five- to tenfold in the myofibers as compared to the myoblasts. Pulse-labeling with [ 3H]uridine and pulsechase experiments with either [ 3H]uridine or [ 3H]methyl-labeled methionine, combined with the measurement of the specific activity of the nucleoside triphosphate pool, demonstrate that this decrease in ribosomal RNA accumulation is produced by a similar five- to tenfold decrease in rRNA synthesis in the myotubes. This regulation is primarily at the level of transcription of 45 S precursor rRNA. We have found no evidence for turnover of mature cytoplasmic rRNA, “wastage” of rRNA precursors during processing, or rapid degradation of the newly synthesized 45 S precursor rRNA in L 6E 9 myoblasts or myotubes. We conclude that in L 6E 9 cells the regulation of rRNA accumulation during differentiation is completely accounted for by the measured changes of rRNA synthesis at the transcriptional level.

A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10 −8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA.

Regulation of Ribosomal RNA Accumulation by Auxin in Artichoke Tissue

Artichoke (Helianthus tuberosus L.) tuber tissue cultured in the presence of the auxin 2,4-dichlorophenoxyacetic acid accumulates ribosomal RNA at a rate of 0.135 micrograms per hour per explant whereas there is little accumulation in nontreated tissue. The addition of auxin enhanced the transcription of the 2.5 x 10(6) precursor 3.5-fold and increased the rate of processing 1.8-fold. The major effect of auxin, however, was a vast increase in the rate of processing of the 1.39 x 10(6) precursor to the 1.3 x 10(6) mature ribosomal RNA. The incorporation of label into the 0.7 x 10(6) mature ribosomal RNA of treated tissue was in 10-fold excess over the control after a 30-minute pulse and remained so throughout the remainder of the labeling period. This level, however, was not reached for the complementary 1.3 mature RNA until 3 hours of continuous labeling, decreasing from a initial value of 40-fold excess. A complication in the processing of ribosomal RNA is the apparent increase in the stability of the 0.7 x 10(6) mature RNA with auxin treatment.

Epidermal Growth and Ribosomal RNA Accumulation in Regenerating Mouse Epidermis Following Abrasion

Epidermal regeneration following abrasion of mouse skin can be divided into 2 phases. The first phase consists of a period of massive epidermal production. Epidermal wet weight in a standard area of skin increases over 6 × that of normal within 5 days after abrasion and remains at this level until about the 8th day. Associated with the increase in epidermal wet weight is a marked increase in total epidermal protein and DNA. The second phase, characterized by a period of regression, begins about 8 days after abrasion, during which the epidermis gradually returns to its normal thickness, and there is a gradual return toward normal levels of wet weight, total protein and DNA. Ribosomes accumulate in large numbers in epidermal cells during the period of massive epidermal production, as measured by the increases in rRNA/DNA and rRNA/gm epidermis. Ribosome accumulation reaches a peak of over 5 × that of normal by 5 days after abrasion, remains high for the next 3 days, and during the period of epidermal regression, gradually decreases approaching normal levels by 21 days after abrasion. A hypothesis is presented that the large accumulation of ribosomes, along with the other associated metabolic regulatory changes, acts as a driving force to keep epidermal cells proliferating and thus helps produce the epidermal hyperplasia.

Synthesis of Plastid and Cytoplasmic Ribosomal RNAs in Isolated Pumpkin Cotyledons: I. Effect of Detachment and Light

Summary During the first four days of pumpkin seed germination in darkness both cytoplasmic and etioplast rRNAs accumulate in cotyledons of intact seedlings. The cotyledons isolated from dry seeds can grow without embryonic axis in darkness. The isolation accelerates the rate of both cytoplasmic and plastid ribosomal RNA accumulation in cotyledons during 72 h of their development, in comparison to those of attached etiolated seedlings. Light markedly stimulates both the growth of isolated cotyledons and the increase in amount of the two ribosomal RNAs, but this effect is more pronounced in the accumulation of chloroplast rRNA.