Increase In rRNA Research Articles

Chemotherapy agents reduce protein synthesis and ribosomal capacity in myotubes independent of oxidative stress.

Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side effects associated with CAs promote muscle loss by incompletely understood mechanisms. To address this problem, we first identified how oxidative stress impairs protein synthesis in C2C12 myotubes. Transient elevations in reactive oxygen species (ROS) resulted in protein synthesis deficits and reduced ribosomal (r)RNA levels. Oxidative stress did not reduce rRNA gene (rDNA) transcription, but it caused an increase in rRNA and protein oxidation. To determine whether CAs affect protein synthesis independent of oxidative stress, we exposed myotubes to Paclitaxel (PTX), Doxorubicin (DXR), or Marizomib (Mzb) at doses that did result in elevated ROS levels (sub-ROS). Exposure to CAs reduced protein synthesis and rRNA levels, but unlike oxidative stress, sub-ROS exposures impaired rDNA transcription. These results indicate that although oxidative stress disrupts protein synthesis by compromising ribosomal quantity and quality, CAs at sub-ROS doses compromise protein synthesis and ribosomal capacity, at least in part, by reducing rDNA transcription. Therefore, CAs negatively impact protein synthesis by causing oxidative stress in addition to directly reducing the ribosomal capacity of myotubes in a ROS-independent manner.

Intra-genomic variation in the ribosomal repeats of nematodes.

Ribosomal loci represent a major tool for investigating environmental diversity and community structure via high-throughput marker gene studies of eukaryotes (e.g. 18S rRNA). Since the estimation of species’ abundance is a major goal of environmental studies (by counting numbers of sequences), understanding the patterns of rRNA copy number across species will be critical for informing such high-throughput approaches. Such knowledge is critical, given that ribosomal RNA genes exist within multi-copy repeated arrays in a genome. Here we measured the repeat copy number for six nematode species by mapping the sequences from whole genome shotgun libraries against reference sequences for their rRNA repeat. This revealed a 6-fold variation in repeat copy number amongst taxa investigated, with levels of intragenomic variation ranging from 56 to 323 copies of the rRNA array. By applying the same approach to four C. elegans mutation accumulation lines propagated by repeated bottlenecking for an average of ~400 generations, we find on average a 2-fold increase in repeat copy number (rate of increase in rRNA estimated at 0.0285-0.3414 copies per generation), suggesting that rRNA repeat copy number is subject to selection. Within each Caenorhabditis species, the majority of intragenomic variation found across the rRNA repeat was observed within gene regions (18S, 28S, 5.8S), suggesting that such intragenomic variation is not a product of selection for rRNA coding function. We find that the dramatic variation in repeat copy number among these six nematode genomes would limit the use of rRNA in estimates of organismal abundance. In addition, the unique pattern of variation within a single genome was uncorrelated with patterns of divergence between species, reflecting a strong signature of natural selection for rRNA function. A better understanding of the factors that control or affect copy number in these arrays, as well as their rates and patterns of evolution, will be critical for informing estimates of global biodiversity.

Regulation of rDNA transcription and ribosome biogenesis at the onset of skeletal muscle hypertrophy

We investigated the regulation of ribosomal (r)DNA gene transcription and ribosome production at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris muscle by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription as determined by 45S pre‐RNA abundance paralleled the increase in rRNA content. Increased transcription and protein expression of c‐Myc and its downstream POL I regulon (POL1RB, TIF‐1A, PAF53, TTF1 & TAF1C) was consistent with the increase in rRNA. Other factors involved in rDNA transcription (UBF) and chromatin remodeling (WSTF) were induced by mechanical loading in a similar fashion. Chromatin‐IP revealed that these factors, together with histone H3 lysine 9 acetylation abundance relative to H3 were also enriched at the rDNA promoter. Our results demonstrate that mechanical loading induces ribosome production via epigenetic and transcriptional mechanisms at the rDNA promoter.Supported by the Swedish Research Council (VR) and the Swedish Nat Ctr for Sports Res (CIF)

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from -3 C to -40 or -50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate-requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O(2)-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.

The in vivo synthesis of stable RNA within the vagus nerve following vagotomy

The in vivo synthesis of rRNA and tRNA within the proximal region of the rat vagus nerve, up to and including the crush site, rapidly increased following vagotomy. Synthesis was maximal at 2 days post-crush when a 15-fold increase in rRNA was observed. This increase was localized to the region 2–3 mm either side of the crush site. Apparent DNA synthesis also appeared substantially elevated at 2, 4 and 7 days post-crush. These results are consistent with a Schwann cell hypertrophy at the site of injury which facilitates removal of axonal debri and myelin and provides structures to receive new axonal sprouts.

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [(3)H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.

Selective enhancement of ribosomal RNA synthesis of crown rust-infected oat leaves by stem excision

In leaves of Avena sativa infected by a compatible race of Puccinia coronata avenae, a rapid increase of ribosomal ribonucleic acid (rRNA) was found when the stem was excised 4 days after inoculation, at which time the fungus was in the beginning of differentiation toward uredosorus formation. Concentrations increased up to 1·5-fold within 4 h after excision, while in the attached control the rRNA concentration increased gradually and reached the same level 16 h later. Stem excision did not affect the concentration of soluble RNA (sRNA) and deoxyribonucleic acid (DNA). This selective increase of rRNA by stem excision was observed neither in the infected leaves 3 and 5 days after inoculation, nor in the noninoculated control leaves. Experiments using [ 32P]orthophosphate indicated that the increase of rRNA was due to enhanced rRNA synthesis. No appreciable changes were shown for the synthesis of sRNA, tenaciously bound RNA and DNA. Polyacrylamide gel electrophoretic separation of rRNA species revealed that the increase of rRNA was due to cytoplasmic rRNA and not due to chloroplast rRNA.

Control of RNA level and of RNA ratios in the latex ofHevea brasiliensis Müll. Arg. effect of latex tapping and of growth regulators

The association between latex RNA and latex production was examined using MAK column chromatography techniques. In young untapped trees the introduction of tapping or the treatment of bark with growth regulators resulted in an increase of RNA level and of rRNA/tRNA ratio in the latex. In regularly tapped trees an increase in rRNA but not in tRNA was brought about by increasing the tapping frequency. Treatment with growth regulators had the same effect but essentially only through the related enhancement of latex export from latex vessels. During latex flow, the highest RNA level was registered in latex fractions originating from the most heavily drained areas of bark. Using32P labeling, evidence was obtained that the export of latex results in an enhancement of rRNA migration into the inner latex containing space of the vessels. This is considered as the reason of the generally observed association of high RNA level and of high rRNA/tRNA ratio with high latex yield. It is proposed that in controlling the RNA level and RNA proportions in the latex an important role is played by changes in turgor pressure associated with the loss of latex which may influence the export of RNA from the nucleus through related induction of pressure disequilibrium between the nucleoplasm and the latex cytoplasm.

Free and membrane-bound polyribosomes in normal and Rauscher-virus-infected mouse spleen cells

1. Free and membrane-bound polyribosomes and ribosomal monomers were isolated from normal and Rauscher-virus-infected mouse spleens by means of discontinuous sucrose density gradients. 2. The addition of ribonuclease inhibitor from rat liver was essential to protect these polyribosomes from degradation. To separate the smooth and rough membranes from ribosomal monomers an additional centrifugation step through a continuous sucrose density gradient was necessary. 3. After infection a marked increase in rRNA from both membrane-bound and free polyribosomes was observed. Treatment of the membrane-bound polyribosomes with sodium deoxycholate yielded only 80S particles even when ribonuclease inhibitor was added. 4. A striking feature of the infected spleen was the occurrence of large polyribosomes. Up to 40 monomers per polyribosome could be counted on electron micrographs.