RNA Synthetic Capacity Research Articles

Abstract 4529: The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma

Abstract PI3Kδ is the major PI3K family class I isoform expressed in B-cells that is known to promote cancer cell growth and survival. PI3Kδ pathway is activated through cell surface receptors, e.g. B-cell receptor signaling. This suggests that PI3Kδ is a therapeutic target for B-cell hematological malignancies. Idelalisib is a potent and selective PI3Kδ inhibitor (IC50=19nM) that is currently under investigation in Phase II/III trials in B-cell neoplasms. In solid tumors, it has been established that PI3K/Akt axis regulates transcription and translation processes. Based on the overexpression and hyperactivity of PI3Kδ in B-cell lymphoma such as mantle cell lymphoma (MCL), and PI3Kδ-specific inhibition by idelalisib, we hypothesized that idelalisib treatment will 1) impact the transcription and translation processes that are PI3K/Akt-dependent, 2) disrupt RNA and protein synthetic capacity, and 3) decrease short-lived oncogenic mRNA and proteins levels that are important for MCL cell survival. Idelalisib (0.5 to 5μM) induced modest levels of apoptosis (<15%) in MCL cell lines and primary cells. A reverse-phase protein array (RPPA) study suggested that idelalisib treatment resulted in a decrease in phosphorylation of transcription regulators (GSK-3αβ and c-Myc), and protein synthesis regulators (S6, p70S6K, 4E-BP1, eIF4 and mTOR) in MCL cell lines and primary cells. Immunoblot analysis suggests that a 4hr treatment with idelalisib (0.5 or 1μM) resulted in a decrease of phosphorylated Akt (T308 and S473) levels in both JeKo-1 and Mino cells following IgM stimulation. In parallel, phospho-levels of GSK-3β (S9), 4E-BP1 (S65) and S6 (S235/236) were also decreased. In IgM-stimulated MCL cell lines, moderate to significant decrease in global RNA synthesis (10-15% decrease in JeKo-1, 50% decrease in Mino) and protein synthesis (15% decrease in JeKo-1, >20% decrease in Mino) were detected with 72hr incubation of idelalisib (0.5 to 1μM). Consistent with cell line data, in B-cell lymphoma primary cells, transcription levels were decreased by 20% or >50% following a 24hr treatment with 1 or 5µM idelalisib, respectively; and a 40% decrease of translation level was detected with 5µM idelalisib. In Jeko-1, early response genes MCL1 mRNA level was reduced following 24hr idelalisib treatment, whereas the other mRNAs (PIM2, MYC, CCND1) were decreased to a lesser extent. Mcl-1 and cyclin D1 protein levels were decreased in JeKo-1 and Mino cells following short incubation of idelalisib (0.5 or 1µM). In summary, idelalisib was effective in reducing phosphorylation of Akt and its downstream effectors in MCL cell lines (GSK-3β, 4E-BP1 and S6). Idelalisib inhibits global RNA and protein synthesis, and decreases the levels of short-lived survival proteins. Collectively, these data imply that similar to solid tumors, PI3K regulates key factors involved in gene transcription and protein translation in MCL. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Varsha Gandhi. The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2014-4529

Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket

The rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly.

Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants.

In order to harness RNA replication for the amplification of mRNAs expressed from recombinant vectors and vaccines, we constructed a VV recombinant that expressed the RNA replicase encoded in the larger genomic segment of the nodavirus FHV. When both termini of the VV-derived transcript were correct, the encoded enzyme replicated its own mRNA, and replication dominated the RNA synthetic capacity of the cell. The smaller genomic segment of FHV could also be replicated by the enzyme when supplied in trans, either by coinfection with another VV recombinant or by transfection of an appropriate plasmid. However, two requirements had to be fulfilled for replication of the smaller FHV RNA segment. The first was the prior replication of the larger genomic segment, which was interpreted as a mechanism to achieve sufficient replicase synthesis before the onset of coat protein synthesis. The second was the presence in the smaller genomic RNA of an internal region between about nucleotides 525-620. Work is in progress to elucidate the reasons for these requirements for RNA 2 replication.

News from the nucleolus: rRNA gene expression

Although the typical, actively growing eukaryotic cell contains over 10 000 different transcripts, half of its RNA synthetic capacity is devoted to the production of a single kind of RNA. This is the pre-ribosomal RNA, which is synthesized in a special compartment of the nucleus, the nucleolus, and is the exclusive product of transcription by RNA polymerase I. In vivo and in vitro approaches have revealed the major features of rRNA gene transcription and of the subsequent processing of the primary transcript.

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.

RNA and protein biosynthesis in the fat body of dormant pupae and pharate adult Heliothis zea

1. 1. Fat body RNA and protein synthesis were measured in vivo in Heliothis zea during pupal diapause and metamorphosis to determine the metabolic basis for termination of the “weak” diapause of this species in response to warming temperatures. 2. 2. RNA and protein synthesis declined during the first two weeks of diapause, but RNA synthesis increased during sustained diapause to a level only 36% below that of developing pharate adults. 3. 3. Protein synthesis responded more than RNA synthesis to diapause termination and increased by 5-fold before adult emergence. 4. 4. Maintenance of the RNA synthetic capacity during extended diapause suggests that translational processes are affected most by diapause termination.

In vitro system for middle T4 RNA. I. Studies with Escherichia coli RNA polymerase.

We describe crude in vitro systems from T4-infected cells which reflect in vivo T4 regulation. Lysates from cells which had been infected in the presence of chloramphenicol manifest the same polarity of RNA synthesis as did the infected cells. Next, we describe a complementation system between lysates which have no RNA synthetic capacity and purified RNA polymerase; in this system, delayed early RNA synthesis in vitro depends on the presence of an active mot gene product. Mot activity controls middle mode gene expression in vivo. In vitro, not activity in the lysate directs RNA polymerase to initiate on regions of DNA that are otherwise inaccessible. This mot-dependent delayed early RNA synthesis in vitro is seen at 0.1 and 0.2 M KCl, but not at 0.05 M KCl. We present a model in which mot is a DNA melting protein necessary for recognition of a middle promoters by either Escherichia coli or T4-modified RNA polymerase which contains E. coli sigma subunit.

Noncytopathic mutants of Newcastle disease virus are defective in virus-specific RNA synthesis.

We have studied virus-specific RNA synthesis in cells infected by six noncytopathic (nc) mutants of the Australia-Victoria wild-type strain (AV-WT) of Newcastle disease virus (NDV) (19). The rates of NDV-specific RNA synthesis in mutant infection were three to sevenfold lower than those observed in wild-type infection. Velocity sedimentation of this NDV-specific RNA revealed that the lower rates of synthesis in mutant infection correlated with reduced accumulation of 18S and 35S mRNA. Electrophoresis in polyacrylamide-urea gels showed that accumulation of all of the 18S mRNA species was reduced and no new species could be detected. Primary transcription appeared unaltered in mutant infection. Cells infected with two naturally occurring avirulent strains of NDV also showed less accumulation of 18S mRNA. Electrophoresis of this RNA resulted in patterns which differed from those obtained with RNA from either AV-WT or nc mutant infection. Complementation for RNA accumulation between the nc mutants and RNA- temperature-sensitive mutants of AV-WT (32) suggested a common defect in the nc mutants. Analysis of plaque-forming revertants of five of the nc mutants revealed that viral RNA synthetic capacity, cell killing, and plaque-forming ability correlated absolutely. These results suggest that viral RNA synthesis and cytopathogenicity may be causally related. In addition, several of the plaque-forming (and cell-killing) revertants were found to be unable to induce fusion from within in infected cell cultures. This result, coupled with the finding that several of the nc mutants are capable of wild-type levels of fusion from within, suggests that the ability to cause such fusion does not correlate with the ability to kill cells.

Interaction of host-coded and virus-coded polypeptides in RNA phage replication.

The enzymes responsible for replication of the RNA of the single-stranded RNA bacteriophages contain, in addition to one phage-coded polypeptide, three host-coded polypeptides taken from the protein biosynthetic machinery: ribosomal protein S1 and the elongation factors Tu and Ts. While S1 performs a function in RNA replication derived from its protein synthetic function, mRNA binding, the reactions catalysed by the elongation factors in protein synthesis are apparently dispensible for RNA replication. In the replicase, these polypeptides, acting as the EF-Tu . Ts complex, play a fundamental structural role. Replacement of the endogenous EF-Tu with mutant EF-Tu, itself stable, causes the RNA replicase to become unstable. The possibility that EF-Tu . Ts is solely a structural protein in the RNA replicase is suggested by experiments showing that a variety of modifications of the elongation factors can be tolerated without loss of RNA synthetic capacity. In fact, EF-Tu . Ts from distantly related bacterial species can substitute for E. coli EF-Tu . Ts in RNA replicase. Evidence is presented that the high in vitro template specificity of Q beta replicase may be accomplished through modulation of the level of GTP required for initiation of transcription. Different natural and synthetic RNAs require quite different GTP concentrations. Mn2+ ions, which extend the range of templates transcribed by Q beta replicase, lower the requirement for GTP. High ionic strength, which alters the conformation of Q beta replicase such that template specificity is increased, raises the GTP requirement. An additional host coded protein required for in vitro Q beta RNA replication, host factor (HF), interacts specifically with Q beta RNA. This polypeptide acts by allowing Q beta replicase to initiate RNA synthesis with Q beta RNA at reduced GTP concentration.

Developmental restrictions on hormone modulated gene transcription. I. Effects of auxin on template capacity and chromatin-bound RNA polymerase

The auxin induced growth response of the developing soybean hypocotyl has been employed as a model system to investigate the transcriptional mechanisms which may in part determine this tissue's hormonal responsiveness during early differentiation. Thus, the molecular components present at the responsive (4 day old) developmental stage, but absent at the unresponsive (8 day) stage, are regarded here as developmental “off-switches” which may commit the older tissue to the synthesis of a limited ensemble of gene products. Such commitments are considered central to the onset of the terminal developmental stages of maturity and senescence. The initial studies have focused on the RNA synthetic capacity of chromatin isolated from control and auxin-induced 4 and 8 day old seedlings. While the young tissue displayed a 160% increase in chromatin bound RNA polymerase activity in vitro, the older plant showed only an 18% increase in this parameter following auxin treatment. Investigations with an exogenous source of RNA polymerase ( Escherichia coli) ruled out template inaccessibility to RNA polymerase as a limiting component in 8-day unresponsive chromatin. In addition, new hormone induced transcripts as determined by nearest neighbor analysis of the RNA synthesized in vitro were detected if the reaction was templated by 4-day hormone induced chromatin and its bound RNA polymerases. However, auxin specific transcripts were not observed with 8-day induced chromatin unless the reaction was supplemented with an exogenous RNA polymerase. The findings reported herein suggest that limitations on hormone controlled gene transcription may in part be determined at the RNA polymerase level, and suggest a developmentally programmed change in the specificity of the endogenous RNA polymerase enzymes.

Cytosol-hormone stimulation of transcription in the aquatic fungus, Achlya ambisexualis

Incubation of Achlya chromatin with antheridiol and the cytosol from male strain E87 increases the RNA synthetic capacity of the chromatin. Neither cytosol, nor antheridiol alone, nor mixtures of hormone with cytosol from the female strain 734 stimulated transcription of the chromatin. A temperature-dependent incubation of the steroid-cytosol complex and high ionic strength (0.05 – 0.12 M potassium chloride) was required for optimal hormone-cytosol stimulation of in vitro transcription. These results strongly suggest that complex hormonal control mechanisms similar to those found in animal systems exist in the primitive fungus Achlya.

Nucleolar RNA synthesis in the liver of partially hepatectomized and cortisol-treated rats

RNA synthesis by isolated nucleoli from rat liver is significantly enhanced 12–14 h after partial hepatectomy and 4 h after cortisol administration. The increased RNA synthetic capacity is demonstrable also in the respective high salt nucleolar extracts and in Biogel A-1.5 filtration fractions of the nucleolar extracts. DNA saturation experiments using nucleoli and Biogel fractions from control and treated animals as RNA polymerase source, have demonstrated, that independent of the extent of RNA synthesis, saturation of transcription is reached at the same concentration of exogenous template. We conclude that the activity and not the amount of nucleolar RNA polymerase is increased as a result of partial hepatectomy or cortisol administration. Parallel to the effects on RNA polymerase, the activity of RNA-degrading enzymes present in nucleoli is also enhanced by the same treatment.

Changes in Histone Acetyl Content and in Nuclear Non-Histone Protein Composition of Avian Erythroid Cells at Different Stages of Maturation

Abstract Duck erythroid cells were fractionated by centrifugation in bovine serum albumin density gradients. Differences in cell density were correlated with the stage of maturation as determined by biochemical and morphological parameters. Avian erythroid cells increase in density as they mature. Cell populations corresponding to known stages in differentiation were compared with regard to histone composition and degree of acetylation, nature and amount of the non-histone nuclear proteins, and DNA and RNA synthetic capacities. Although the total histone to DNA ratio of the chromatin does not vary appreciably during maturation, the relative proportion of the erythroid cell-specific histone F2c increases at least 2-fold with the increasing age of the cell, whereas the other histones, except F2a2, decrease. The degree of acetylation of histones F2a1 and F3 decreases with the increasing age of the cell. The non-histone protein complement of the erythroid cell nucleus decreases 6-fold during maturation. Analysis of the non-histone proteins in polyacrylamide gels containing sodium dodecyl sulfate shows a limited heterogeneity and a remarkable similarity in their electrophoretic patterns at different stages in maturation. Quantitative rather than qualitative changes predominate as maturation proceeds. The decline in non-histone protein content with age is striking particularly in the lower molecular weight components. However, a protein band of molecular weight 55,000 is conserved selectively during maturation and it represents the predominant component of the non-histone nuclear proteins of the mature erythrocyte. The nucleoprotein changes occurring during erythrocyte maturation parallel a decrease in the amount of chromatin-associated RNA and the rate of RNA synthesis. The average RNA synthetic capacity of the isolated erythroblasts and early polychromatic erythrocytes is about 8 times higher than that of the mature erythrocyte fraction.

Studies on the polyribosome-associated RNA in BHK21 cells infected with Semliki Forest virus

We have developed a method that allows us to isolate with high yield RNP particles containing intact RNA from BHK21 cells infected with Semliki Forest virus. Two types of virus-specific RNA sedimenting with about 42 S and 26 S on sucrose gradients are found in the gradient fractions containing the large and intermediate-sized polyribosomes. That the 26 S RNA and at least 50% of the 42 S RNA are presumably part of the polyribosomes has been shown by CsCl density gradient analysis of the fixed ribonucleoprotein particles. We suggest therefore that both molecules are virus specific mRNA. From the quantitative aspects of our results, we conclude that they are the main species of virus specific polyribosome associated RNA in the infected cells. The total virus-specific RNA synthesized during different 2 hr intervals, which is incorporated into polyribosomes, is greater than 20% during the 0 to 2 hr p.i. interval and decreases later. We suggest therefore that especially during the first hours after infection, a major part of the total RNA synthetic capacity of the virus is directed toward mRNA production. Both polyribosome-associated molecules do not hybridize to RNA extracted from the SFV particle.

Increased RNA synthesis in nuclei isolated from rat liver tissue slices incubated with cyclic adenosine 3′,5′-monophosphate or glucagon

Incubation of rat liver tissue slices with cyclic adenosine 3′,5′-monophosphate (cyclic AMP) for 30 min results in a dose-dependent increase in RNA synthesizing capacity of nuclei prepared from these slices, with a doubling of synthetic rate observed at 10 −7 m cyclic AMP. The cyclic AMP effect is observed when RNA polymerase activity is measured either in the presence of Mg 2+ and low ionic strength, or Mn 2+ and high ionic strength. Experiments employing saturating amounts of exogenous bacterial RNA polymerase suggest that the cyclic AMP-induced stimulation occurs primarily at the level of template activity. Other cyclic nucleotides tested in the same manner are ineffective in stimulating RNA synthesis by tissue slice nuclei. In addition to cyclic AMP, adenosine 5′-monophosphate (5′-AMP) consistently produced small increases in nuclear RNA synthesis although never of the magnitude seen with the cyclic nucleotide. An increased capacity for RNA synthesis is also seen in nuclei isolated from liver slices incubated with glucagon at concentrations from 0.5 μg/ml to 50 μg/ml. A maximal stimulation of approximately twofold occurs at a glucagon concentration of 1.0 μg/ml. Liver slices incubated with optimal concentrations of cyclic AMP and glucagon simultaneously show that the effects of the two agents on RNA synthesis are not additive. The results indicate that cyclic AMP at physiological concentrations can stimulate RNA synthetic capacity in vitro, and that the effect mimics a similar response to glucagon. Since it is known that glucagon causes an increase in liver concentrations of cyclic AMP and a subsequent induction of some liver enzymes, it is suggested that cyclic AMP-mediated control of RNA synthesis may be involved in such regulation.

Reversal of inhibition of membrane-bound RNA synthesis in Escherichia coli infected with R23

1. Infection by RNA bacteriophage R23 causes a release of ribosomes from a membrane-DNA-RNA polymerase complex isolated from Escherichia coli. This release may be responsible for the reduction in RNA synthetic capacity seen after phage infection. 2. The addition of ribosomes to such a membrane preparation restores the RNA synthetic capacity to uninfected levels. The ribosomes may exert their effect by binding to nascent RNA chains. 3. Exogenous RNA polymerase stimulates RNA synthesis by the membrane complex under conditions which probably prevent the initiation of new RNA chains. The mechanism of its effect may be analogous to that of ribosomes in this system.

In Vitro Synthesis of Ribosomal Ribonucleic Acid by a Deoxyribonucleic Acid-Protein Complex Isolated from Escherichia coli

Abstract A DNA-RNA polymerase complex has been isolated from EDTA-treated Escherichia coli MRE 600. This fraction contains virtually all of the RNA synthetic capacity expressed in the EDTA-treated cells. The in vitro synthesis is sensitive to rifampicin, indicating that the complex is actively initiating RNA chains. Analysis of the product RNA by hybridization competition shows that between 20 to 30% is rRNA. Guanosine tetraphosphate (ppGpp) inhibits global RNA and rRNA synthesis equally, and no preferential inhibition of rRNA synthesis is detected.

Absorption, accretion and endogenous faecal excretion of calcium in the newborn infant.

IT is known that the metabolism of the non-histone, acidic proteins associated with DNA in the chromatin of animal cells is influenced by steroid hormones. The effects are often highly specific; for example, the administration of hydrocortisone to adrenalectomized rats leads to a selective stimulation of synthesis of a liver chromosomal protein of molecular weight 41,0001. Hydrocortisone also alters the pattern of phosphorylation of chromosomal proteins in the liver, differential effects becoming evident within minutes after injection of the hormone2,3. Similar specific alterations in the synthesis or phosphorylation of nuclear proteins have been observed in uterine cells responding to oestrogens4, in kidney cells following aldosterone administration (C. Liew, personal communication), in prostate cells stimulated by androgens5, and in insect chromosomes stimulated by ecdysone6. In all these cases, the steroid hormone also affects the RNA synthetic capacity of the target cells, presumably as a result of altered template capacity of the modified chromatin.

Estrophile to nucleophile in two easy steps

A variety of self-consistent experimental evidence supports the concept that estrogcnic hormones interact with their target tissues by a multi-stage mechanism in which the hormone first associates with the 4S binding unit of an 8S extranuclear receptor protein, activating it to undergo conversion to a 5S form. This temperature-dependent transformation takes place when the receptor is bound to estradiol, but not when it is uncomplexed or bound to estrone. The transformed hormone-receptor complex migrates to the nucleus where it associates with an acceptor site in the chromatin. Analytical amounts of the 5S nuclear complex and a calciumstabilized 4S cytosol complex have been isolated from calf uterus in apparently pure state, opening the way for the large scale purification of these different forms of the receptor protein. The estrogen-receptor interaction sequence appears to be involved in the acceleration of biosynthetic reactions in hormone-dependent tissues. Exposure of isolated uterine nuclei either to transformed complex or to native complex under conditions where transformation can take place causes a significant increase in their ability to synthesize RNA. Similar treatment does not enhance the already high RNA synthetic capacity of liver or kidney nuclei. It is suggested that an important function of the hormone is to promote the conversion of the receptor protein to an active form which can enter the nucleus and alleviate a defficiency in RNA synthesis, characteristic of estrogen-responsive tissues.

Tissue-specific stimulation of RNA synthesis by transformed estradiol-receptor complex

Exposure to the estradiol-receptor complex of uterine cytosol increases the RNA synthetic capacity of purified uterine nuclei but not of liver or kidney nuclei. This stimulation is effected only by complex in which the binding unit of the receptor has undergone estrogen-induced conversion from the native (4S) to the transformed (5S) form. It is suggested that an important function of estrogenic hormone is to promote transformation of the receptor protein to an active form which can enter the nucleus and stimulate RNA synthesis.