Controlling nuclear RNA levels.

RNA turnover is an integral part of cellular RNA homeostasis and gene expression regulation. Whereas the cytoplasmic control of protein-coding mRNA is often the focus of study, we discuss here the less appreciated role of nuclear RNA decay systems in controlling RNA polymerase II (RNAPII)-derived transcripts. Historically, nuclear RNA degradation was found to be essential for the functionalization of transcripts through their proper maturation. Later, it was discovered to also be an important caretaker of nuclear hygiene by removing aberrant and unwanted transcripts. Recent years have now seen a set of new protein complexes handling a variety of new substrates, revealing functions beyond RNA processing and the decay of non-functional transcripts. This includes an active contribution of nuclear RNA metabolism to the overall cellular control of RNA levels, with mechanistic implications during cellular transitions.

Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS.

Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about ten times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)(+) mRNA were exported to the cytoplasm, where DPR proteins were produced at >100-fold higher level than in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production, but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.

The cortical subventricular zone-specific molecule Svet1 is part of the nuclear RNA coded by the putative netrin receptor gene Unc5d and is expressed in multipolar migrating cells.

Although Svet1 RNA is a widely used marker for the subventricular zone (SVZ) of the embryonic cerebral cortex, its function remains completely unknown. We report finding that Svet1 contains a high proportion of repetitive sequences and maps in the first intron of the putative Netrin receptor gene Unc5d. The direction of transcription of Svet1 is the same as that of Unc5d. The Svet1 RNA was detected in the nucleus but not in the cytoplasm. Both Svet1/Unc5d RNAs and UNC5D protein were localized in the multipolar cells in the SVZ throughout cortical development. These results suggest that Svet1 RNA is part of the sequence of the primary transcript of Unc5d in the nucleus that is spliced out before the mRNA is transported to the cytoplasm. Thus, the previously reported "SVZ-specific expression of the Svet1 RNA" in fact indicates putative involvement of UNC5D signaling in the multipolar migrating cells.

[Investigation of properties of the thermostable protein complex obtained from the myocardium left ventricles of adult white rats].

A thermostable protein complex (TSPC) obtained from the myocardium ventricules of adult rats inhibits mitotic and transcriptional activity of cardiomyocytes. At the same time this complex is more active on early stages of the postnatal ontogenesis in rats, aged 1 and 5 days. Following its action on RNA synthesis, the TSPC reveals tissue specificity only in cells with terminal differentiation, and is determined by its nuclear membranes. We continue studies for identifying the molecular weight and chemical nature of the TSPC, and the role of its different fractions in regulation of proliferation processes. Besides, it is planned to produce antibodies against TSPC fractions with the purpose to block its inhibitory effect on myocyte regeneration in the damaged myocardium.

Evidence for the preventive effect of the polyunsaturated phytol side chain in tocotrienols on 17beta-estradiol epoxidation.

BACKGROUND: We found that 17beta-estradiol (E2) could be activated by epoxidation to bind DNA and to inhibit nuclear RNA synthesis. Vitamin E compounds are powerful antioxidants and chain-breaking free radical scavengers. The chromanol ring in Vitamin E is believed to be involved in these reactions. METHODS: Here, we examined the preventive effect of alpha-tocopherol, alpha-, gamma- and delta-tocotrienols on E2 activation. RESULTS: We found that when any one of these Vitamin E compounds was mixed with E2 for epoxidation by the epoxide-forming oxidant dimethyldioxirane (DMDO), alpha-tocopherol was the least effective as compared with the tocotrienols against the formation of E2 epoxide as reflected by the loss of the ability of E2 to inhibit nuclear RNA synthesis. This conclusion was further confirmed by the binding studies of [3H] labeled E2 to DNA using either DMDO or liver microsomes activation system. CONCLUSIONS: Since the chromanol ring is shared by both tocopherols and tocotrienols and the only difference between these two subgroups of Vitamin E is the phytol side chain, we conclude that the polyunsaturated phytol group in tocotrienols plays a key preventive role in E2 epoxidation. This is the first report showing that the polyunsaturated phytol side chain in tocotrienols is involved in an antioxidative activity and it may also have a preventive effect against the E2 epoxide induced breast cancer carcinogenesis at the initiation.

Developmental regulation of EVF-1, a novel non-coding RNA transcribed upstream of the mouse Dlx6 gene.

We previously reported that sonic hedgehog (Shh) induces the differentiation of rat ventral forebrain neurons expressing a novel marker, EVF-1 [Development 125 (1998) 5079]. In this report, we show that EVF-1 is a novel, developmentally regulated, non-coding RNA, with no homology to other known non-coding RNA sequences. Sequence analysis, in vitro translation, and comparison of the rat and mouse EVF-1 sequences suggest that EVF-1 contains no protein coding regions. Chromosomal location indicates that EVF-1 maps adjacent to the Dlx6 gene on mouse chromosome 6. RNA in situ hybridization of the embryonic rat forebrain shows that EVF-1 is expressed by immature neurons in the subventricular zone and its expression decreases during forebrain development. Whole mount in situ hybridization shows that EVF-1 is expressed at high levels in the branchial arches, ventral forebrain, olfactory bulb, and limbs. EVF-1 expression is linked to Shh and the Dlx family of proteins, genes with a demonstrated importance to ventral forebrain and craniofacial development.

[Effect of human fetal liver cells on the rate of DNA and nRNA synthesis in regenerating rat liver]. / Vliianie émbrional'nykh kletok pecheni cheloveka na sintez DNK i iaRNK v regeneriruiushchei pecheni krys.

The model of partial hepatectomy was utilized to investigate the influence of human fetal liver cells (FLCs) and of human embryo tissue (PCF) postnuclear cytoplasm fraction injection on the rate of DNA and nRNA synthesis in regenerating rat liver cells. Single infusion of FLCs and PCF into the spleen pulp of rats has been shown to increase the DNA synthesis 24 hours after the operation in 2.5 +/- 0.4 and 3.2 +/- 0.5 times respectively. A group of rats has been identified with no influence of the infusion on the DNA synthesis 24 hours after partial hepatectomy, this process having been even retarding in 48 hours after the operation. Meanwhile the FLCs and PCF infusion enhanced the intensity of nRNA synthesis in 72 hours after the operation in all the animal groups. The effect demonstrated is probably caused by the biologically active substances contained in the fetal tissues.

Regulation of Cyp1a1 induction by dioxin as a function of cell cycle phase.

Analyses of CYP1A1 mRNA were used to monitor the responsiveness of murine hepatoma 1c1c7 and human monocytic U937 cells in different phases of the cell cycle to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Concentrations of TCDD capable of inducing CYP1A1 were not cytostatic to either cell line. Steady-state CYP1A1 mRNA contents were reduced (45-90%) in TCDD-treated cultures arrested in G2/M as a consequence of exposure to microtubule disrupters (Colcemid, estramustine, vinblastine) or the microtubule stabilizer Taxol, relative to TCDD-treated asynchronous 1c1c7 cultures. The accumulation of mRNAs corresponding to Nmo1, another TCDD-inducible gene of the Ah battery, was also reduced in TCDD-treated G2/M cultures. Quantitative reverse transcriptase-polymerase chain reaction analyses of CYP1A1 heterogeneous nuclear RNA (hnRNA) revealed that Cyp1a1 transcription was suppressed in G2/M cells. This suppression reflected neither changes in the relative content of the proteins comprising the aryl hydrocarbon receptor (AHR) complex nor a suppression of AHR activation and translocation to the nucleus. Release of 1c1c7 cultures arrested in G2/M restored TCDD responsiveness. Centrifugal elutriation of TCDD-treated asynchronously growing U937 cells was used to prepare populations of cells in specific phases of the cell cycle. Within 3 h of TCDD exposure late G1/early S phase cells had CYP1A1 mRNA contents approximately 1.4- and 3-fold higher than the contents of asynchronous/early G1 and G2/M cultures, respectively. These studies suggest that the transcriptional activation of members of the Ah battery by TCDD is cell cycle-dependent, and markedly suppressed in G2/M cells.

Role of M2 domain residues in conductance and gating of acetylcholine receptors in developing Xenopus muscle.

1. The contributions of specific residues in gamma- and epsilon-subunits to the developmental changes in conductance and open time of Xenopus muscle acetylcholine receptors (AChRs) were investigated. This study was directed primarily at residues in the M2 domains of gamma- and epsilon-subunits; however, the results of additional mutations in the extracellular region flanking M2 and in the amphipathic region between M3 and M4 are also described. 2. The M2 domains of gamma- and epsilon-subunits differ at only three amino acid residues, two of which are adjacent to each other and located near the narrowest part of the pore. These two residues (NI in gamma, SV in epsilon) were found to be major determinants of the difference in conductance and open time of AChRs bearing gamma- or epsilon-subunits. 3. Mutation of N to S in the gamma-subunit converted the long open time of receptors bearing the gamma-subunit (gamma-AChRs) to the brief open time characteristic of receptors bearing an epsilon-subunit (epsilon-AChRs). Conversely, epsilon-AChRs with SV mutated to NI in the epsilon-subunit exhibited a long open time characteristic of gamma-AChRs. 4. Mutation of N to S in the gamma-subunit increased the conductance of gamma-AChRs but did not confer the full conductance of wild-type epsilon-AChRs. Conversely, mutation of SV to NI in the epsilon-subunit reduced the conductance of epsilon-AChRs, but not completely to the level of wild-type gamma-AChRs.

Specific telomerase RNA residues distant from the template are essential for telomerase function.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Differential regulation of two genes implicated in fish reproduction: vitellogenin and estrogen receptor genes.

In rainbow trout as well as in other species, variability of estrogen receptor (ER) gene expression according to the cell type and the physiological state reflects a differential cell and gene sensitivity to estrogen. We previously demonstrated that expression of the rainbow trout estrogen receptor (rtER) and vitellogenin (Vg) genes were induced differently by estrogens in rainbow trout liver. Therefore, these two genes offered a suitable model to study the influence of ER concentration on gene transcriptional activities. In the present study we show that the transcription rate of rtER and Vg genes during an estrogenic treatment are affected differently by variation of cellular ER concentration. We demonstrate that rtER gene exhibits a low threshold response to loaded estrogen receptor, and increasing ER amounts do not affect the transcriptional response of this gene during an estrogenic stimulation. On the contrary, Vg gene expression requires the presence of a higher loaded estrogen receptor level to be induced, and its transcriptional response is directly proportional to the amount of synthesised ER.

[Liver nuclear phospholipids of rats with varying thyroid status]. / Fosfolipidy iader pecheni krys s razlichnym tireoidnym statusom.

Thin-layer chromatography was used for fractionation of rat liver nuclear phospholipids after in vivo administration of 14C-sodium acetate. The administration of T3 to thyroidectomized rats caused a sharp increase in the incorporation of the label in all phospholipids of the nuclear fraction. The action of sphingomyelin and sphingomyelase on RNA-polymerase of nuclei isolated from the liver of thyroidectomized rats was tested. Sphingomyelin was shown to cause stimulation of RNA nuclear synthesis; parallel incubation with sphingomyelinase eliminated a stimulating effect of this phospholipid.

Methylated cap structures in eukaryotic RNAs: structure, synthesis and functions.

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.

Autoradiographic study of 3H-uridine incorporation into nucleoli and DNA/3H-5S DNA in situ hybridization in root nodules and uninfected Lupinus luteus L. tissues.

The autoradiographic method was used to compare the 3H-uridine incorporation and the number of hybridization sites with 3H-5S DNA in yellow lupin root apical meristem, root hair of uninfected roots as well as in root nodule cortex and bacteriod-containing tissue. It has been shown that the number of hybridization sites is proportional to the ploidy level, but not to rRNA synthesis, which is most intense in root apical meristem and young bacteriod-containing cells.

[Influence of the active compounds isolated from pilose antler on syntheses of protein and RNA in mouse liver].

The polyamines of pilose antler (PASPA) consist of putrescine (PU, 70.9%), spermidine (SPD, 26.3%) and spermine (SP, 2.8%). The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver tissue were increased when PASPA was given orally to mice at the dose of 30 mg/kg for 4 successive days. The incorporations of [3H] leucine into liver protein and [3H] uridine into the cytosolic and nuclear RNA were also increased by treatment with PU (21 mg/kg). In addition, the RNA polymerase activity in the solubilized liver nuclear fraction of PU (21 mg/kg)-treated mice was increased. SPD only promoted the synthesis of protein in mouse liver tissue at the dose of 8 mg/kg. However, SP showed no effect on the synthesis of protein and RNA polymerase activity under the used dose (1 mg/kg). The results suggest that PASPA is the main active substance responsible for the promotion of the synthesis of protein and RNA in mouse liver.

Chromatin structure and transcriptional regulation of human papillomavirus type 18 DNA in HeLa cells.

Mapping analysis of the nucleosomal organization of integrated human papillomavirus type 18 (HPV18) DNA in HeLa cells reveals a very prominent nuclease-hypersensitive site within the viral noncoding regulatory region that harbors transcriptional control sequences and coincides with most of the 5' ends of the cytoplasmic early mRNAs. Moreover, it is shown that the conserved coamplified 5' cellular flank, common to all HPV18 copies in HeLa cells and located close to the virus-cell integration site, also contains several distinct hypersensitive sites, accessible not only to DNase I but also to restriction enzymes. Nuclear run-on analysis in isolated HeLa nuclei demonstrates the occurrence of nascent transcripts covering the cellular flank (the late and the viral noncoding regulatory region), indicating that a cellular promoter, marked by the hypersensitive sites, cooperates with the viral control region in generating the HPV18 transcripts. Cycloheximide treatment of HeLa cells results in a reduction of the cytoplasmic steady-state level of the 3.5-kb mRNA corresponding to the viral E6, E7, and parts of the E1 open reading frames (ORFs), whereas the expression of the 1.6-kb transcript corresponding only to the E6 and E7 ORFs is not influenced. Nuclear run-on analysis carried out after the cycloheximide chase reveals that the distribution of nascent transcripts spanning the viral E6, E7, and parts of the E1 region is substantially decreased. In contrast to this finding, an even, pronounced increase of the elongation rate of those transcripts, which cover the cellular flank, the late and the viral noncoding regulatory region was noted indicating a different involvement of regulatory factors in the activity of both promoters.

Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo.

It is well known that actinomycin D, a carcinogen, inhibits DNA-dependent RNA synthesis. The interpretation of this inhibition has been that this carcinogen binds specifically to the deoxyguanosine moiety of a DNA molecule, and thus blocks the template function. This paper presents evidence which suggests that this single mechanistic interpretation of actinomycin D action may not be adequate in eukaryotic cells. Thirty minutes after actinomycin D injection (250 microg/100 g body weight), rat liver nuclear RNA synthesis was inhibited by 81% and nucleolar RNA synthesis was inhibited by 98%. In order to determine whether this inhibition is due to an inhibition of DNA template function or of the RNA polymerase activity, the total nuclear free and engaged RNA polymerases were solubilized and the individual RNA polymerase species were partially purified by DEAE-Sephadex column chromatography. It was found that while the overall enzyme activities of RNA polymerase I and III were not affected, there was a selective inhibition of RNA polymerase II activity (42%). This result suggests that actinomycin D, like aflatoxin B1 and N-hydroxy-2-acetylaminofluorene, inhibits nuclear RNA synthesis at multiple sites; it inhibits nucleolar RNA synthesis by blocking the nucleolar DNA template function, and it inhibits messenger RNA synthesis by inhibiting at least partially the enzyme RNA polymerase II activity per se.