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PubMed2002

Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.

Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.

Apoptosis↗
PubMedDate not supplied

[Effect of low-molecular nuclear RNA on RNA synthesis in isolated nuclei].

An attempt was made to elucidate possible participation of low molecular weight nuclear RNA's (LMWN RNA's) in the transcription process. For this purpose, we studied the effect of individual fractions of LMWN RNA's, isolated by polyacrylamide gel electrophoresis, on the endogenous RNA synthesis in isolated nuclei. We have found no influence of LMWN RNA's on the incorporation of labeled precursors in the acid-insoluble material under the conditions when RNA polymerase I is predominantly active. The results obtained thus indicate that LMWN RNA's do not participate in the regulation of 45S pre-rRNA synthesis and they do not belong to limiting factors in pre-rRNA synthesis.

Adenosine Monophosphate↗
PubMed1978

Sequence of a RNA templated by the 3'-OH RNA terminus of defective interfering particles of vesicular stomatitis virus.

We have sequenced the endogenous RNA polymerase product produced by disrupted purified virions of vesicular stomatitis virus defective interfering particles by using the newer one-dimensional rapid gel sequencing techniques and confirming this with a modified two-dimensional gel vectoring technique. The sequence of this 46-nucleotide RNA is: 5'(pp)pACGAAGACCACAAAACCA-GAUAAAAAAUAAAAACCACAAGAGGG(U)COH3'. We infer that this sequence is identical to the sequence at the 5' end of infectious vesicular stomatitis virus RNA and is complementary to the sequence of the 3'-OH terminus of this defective interfering particle genome RNA.

Base Sequence↗
PubMed2006

Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3' UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3' UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

3' Untranslated Regions↗
PubMed1997

Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes.

BACKGROUND: The oskar (osk) gene encodes a determinant of posterior identity in Drosophila, and the localization of osk RNA to the pole plasm at the posterior pole of the oocyte is essential for development of the embryo. The mechanisms by which osk RNA is localized are unknown. RESULTS: To study the mechanisms underlying localization of osk RNA, we have injected fluorescently labelled RNA into oocytes at stages 9, 10 and 11. Injected osk RNA localizes to the pole plasm, reproducing localization of the endogenous RNA. In oocytes at stages 10 and 11, the long-range movement of injected osk RNA is promoted by a vigorous, microtubule-dependent cytoplasmic flow, or ooplasmic streaming. Treatment with colchicine, a microtubule-destabilizing drug, inhibits ooplasmic streaming and prevents localization of the RNA from an injection site distal to the posterior pole. If the RNA is injected close to the posterior pole, however, it localizes even in the presence of colchicine. Similarly, in small oocytes, such as stage 9 oocytes, localization of injected osk RNA is insensitive to colchicine. CONCLUSIONS: These results reveal that microtubule-dependent cytoplasmic flows could contribute to the long-range transport of osk RNA, whereas microtubule-independent processes could mediate short-range transport. These results also highlight the role of the osk RNA anchor in the localization process.

Animals↗
PubMed1993

Translocation of repetitive RNA sequences with the germ plasm in Xenopus oocytes.

Xlsirts are a family of interspersed repeat RNAs from Xenopus laevis that contain from 3 to 13 repeat units (each 79 to 81 nucleotides long) flanked by unique sequences. They are homologous to the mammalian Xist gene that is involved in X chromosome inactivation. Xlsirt RNA appears first in the mitochondrial cloud (Balbiani body) in stage 2 oocytes and is then translocated as island-like structures to the vegetal cortex at early stage 3 coincident with the localization of the germ plasm. Exogenous Xlsirt RNA injected into oocytes translocates to the location of the endogenous RNA at that particular stage. The Xlsirt RNA repeat sequences are required for translocation and can cause the translocation of heterologous unique RNAs to the vegetal cortex.

Animals↗
PubMed1974

Encephalomyocarditis virus infection of mouse plasmacytoma cells. II. Effect on host RNA synthesis and RNA polymerases.

The effect of encephalomyocarditis virus infection of MOPC 460 mouse plasmacytoma cells on host RNA synthesis and RNA polymerases was investigated. Consistent with work performed in other virus host systems, rates of RNA synthesis appeared to be inhibited in infected cells, whereas RNA degradation appeared normal. These results were further extended with isolated nuclei, in which distinct RNA polymerase activities could be studied under conditions where problems with RNA turnover and endogenous nucleotide pool sizes were insignificant. Endogenous nuclear RNA polymerase II activity was inhibited early postinfection and at 1 to 2 h prior to endogenous RNA polymerase I plus III activity. However, the solubilized enzymes were fully active with exogenous DNA as template. In fact, the levels of RNA polymerases I, II, and III, isolated from infected cells and nuclei, were indistinguishable from levels in uninfected cells and nuclei at each stage of their partial purification procedure. The chromatographic properties of the enzymes on DEAE-Sephadex were also unaltered. Furthermore, the RNA synthetic activity of these isolated enyzmes, or of nuclei isolated from uninfected cells, was resistant to extracts of nuclei or of cytoplasmic fractions from infected cells. These results are discussed in terms of a possible inhibition of RNA synthesis in vivo at the level of transcription initiation.

Animals↗
PubMed1985

Characterization of human endogenous retroviral envelope RNA transcripts.

We characterized the structure of human endogenous retroviral env RNA transcripts by Northern blot hybridization and cDNA cloning. Polyadenylated 3.0- and 1.7-kilobase env RNAs can be identified in placenta, colon carcinoma, and breast carcinoma cells. We have obtained partial cDNA clones of both size classes of RNAs. Both env RNAs contained putative gp70 coding sequences; the 1.7-kilobase species, however, lacked sequences in the 3' env region which could specify a p15E analog. Both cDNA clones contained in-frame termination codons; thus, neither could encode full-length env proteins.

Amino Acid Sequence↗
PubMed1975

Alterations in chromatin functions during aging in vitro.

Age-associated alterations in the chromatin functions of human diploid cells have been observed. These alterations include: (1) a decline in the rate of histone acetylation; (2) a reduction in the rate of RNA synthesis as measured by (a) the rate of 3H-uridine incorporated into the RNA of young and old cells; (b) comparison of the template activity of isolated chromatin from young and old cells using E. coli RNA polymerase and (c) measurement of chromatin template activity using the endogenous RNA polymerase of young and old cells. It is suggested that the nondividing state of old cells may be the result of the inability to synthesize specific RNA molecules (and perhaps specific proteins) necessary for the cell to continue through the cell cycle.

Acetates↗
PubMed1979

Effect of lucanthone (miracil D) on transcription of ribosomal RNA genes from Tetrahymena in vivo and in vitro.

Addition of lucanthone (1-5 mug/ml) to cultures of Tetrahymena results in a preferential inhibition of the synthesis of ribosomal RNA. Transcriptional studies with isolated nucleoli from Tetrahymena demonstrate that the endogenous RNA polymerases of the r-chromatin (chromatin form of rDNA) do not recognize the normal termination and move into the spacer region distal to the terminator in the presence of lucanthone. This is shown by hybridization of the transcript synthesized in the presence of the drug to restriction fragments of rDNA. Lucanthone seems specific in its action on termination as it does not inhibit the elongation process on the chromatin. Among various DNA-binding drugs tested only lucanthone and proflavine are found to cause repression of the termination. The data obtained suggest that the reduced synthesis of rRNA in lucanthone-treated eukaryotic cells is due to lack of reinitiating RNA polymerases possibly caused by improper termination.

Animals↗
PubMed1981

Excessive ingestion of iodide by the rat during pregnancy and lactation. Effects on ribonucleic acid transcription in the pups brain.

The effects of an excessive ingestion of iodide by the rat during pregnancy and lactation on the transcription of RNA were studied in the brain of the pups at 10, 15 and 20 days of age. Cerebral weight, DNA content and synthesis of heterogeneous RNA were not affected during the period studied. The activity of endogenous RNA polymerase I, alpha-amanitin resistant, measured in isolated cell nuclei was significantly reduced in twenty day old rats. Similar conditions meaningfully diminished body weight at the same age.

Animals↗
PubMed1982

Template structural requirements for transcription in vivo by RNA polymerase II.

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei of Xenopus laevis oocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 mini-chromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

Animals↗
PubMed1975

RNA metabolism of murine leukemia virus II. Endogenous virus-specific RNA in the uninfected BALB/c cell line JLS-V9.

Type C virus-specific RNA sequences of BALB/c endogenous virus were detected in JLS-V9 cells (an uninfected BALB/c derived line) by annealing cell RNA with 3-H-labeled virus-specific DNA. Endogenous viruses used in preparing the 3-H-labeled DNA (mostly xenotropic) was prepared from JLS-V9 cells induced to produce virus with iododeoxyuridine. In whole-cell extracts, two virus-specific RNA species, 38S and 27S, were detected. No 60 to 70S virus-specific RNA was found. The same two species of virus-specific RNA were observed in isolated cytoplasmic RNA and in cytoplasmic RNA selected for polyadenylic acid-containing species by binding and elution from oligo(dT) cellulose. Very little, if any, of the virus-specific RNA was active as messenger RNA on polyribosomes. No virus-specific RNA transcribed from genes coding for the BALB/c endogenous N-tropic virus was detected, since 3-H-labeled DNA prepared from endogenous N-tropic virus did not hybridize measurably with JLS-V9 RNA.

Animals↗
PubMed1979

Specific gene transcription in yeast nuclei and chromatin by added homologous RNA polymerases I and II.

When treated at pH less than 4.5, yeast nuclei or chromatin lose endogenous RNA synthetic activity. This activity is regained by addition of exogenous RNA polymerases. The specificity of transcription in this system by homologous RNA polymerases I and III has been investigated by gel electrophoresis, hybridization analysis, and RNase T1 mapping. Exogenous RNA polymerase I selectively transcribes rRNA genes. The transcription of these genes by polymerase I is 30- and 8-fold more selective than RNA polymerase III and Escherichia coli polymerase holoenzyme, respectively. Exogenous RNA polymerase III synthesized RNAs similar in size to authentic 5 S RNA, 4.5 S pre-tRNA, and 4 S tRNA. Eleven per cent of this RNA is 5 S RNA as determined by hybridization. Neither polymerase I nor E. coli polymerase synthesizes detectable quantities of RNA in this size range. AT1 ribonuclease digestion of 5 S RNA synthesized by exogenous RNA polymerase III acting on acid-treated chromatin gives a fragment pattern corresponding to that of 5 S RNA. Thus, RNA polymerase III transcribes the entire 5 S gene in this system.

Cell Nucleus↗
PubMed1976

In vitro transcription of the tryptophan operon in isolated bacterial nucleoids.

In vitro transcription of the trp operon in isolated nucleoids from Escherichia coli was studied. RNA synthesis in this system occurred primarily as a continuation of transcription which had been initiated in vivo; little or no initiation of new RNA chains was observed. Transcription of the trp operon in nucleoids by endogenous RNA polymerase procedded efficiently and ceases sequentially in the order of the gene sequence within the operon. Under these conditions, no appreciable exonuccleolytic digestion of nascent 3H-RNA was found, though some endonucleolytic cleavage was generally seen. Little or no incorporation of 14C-leucine into polypeptides was observed, inspite of tha fact that considerable number of ribosomes and nascent RNA chains were found attached to the isolated nucleoids. The synthesis of trp mRNA continued in the presence of chloramphenicol or fusidic acid, or under conditions where the rebosomal translocation factor G was inactivated. From these and other kinetic studies of trp mRNA synthesis in nucleoids obtained from nonsense strong polar mutants of the trp operon, it was shown that transcription in nucleoids was not connected functionally with transloational processes and thus unable to exhibit polarity effected by a nonsense mutation or by general translational blockage. In studies employing nucleoids from nonsense strong polar mutants of the trp operon, it was demonstrated that RNA polymerase are scantily distributed over the region downstream from the nonsense mutation site of the operon, thereby supporting a notion that in vivo transcription is eventually terminated near the nonsense mutation.

DNA, Bacterial↗
PubMed1988

The role of JH III and ecdysterone in the regulation of the left colleterial gland activities in Periplaneta americana during the reproductive cycle.

We studied the in vitro effects of JH III and ecdysterone on RNA synthesis and secretory activities of the left colleterial gland (LCG) in mature females of Periplaneta americana during the reproductive cycle. We found that RNA synthesis is a cyclic and highly dynamic process. The major increases in poly(A)- RNA synthesis occurred 8 hr before the peak leucine incorporation into colleterial polypeptides, whereas high poly(A)+ RNA synthesis coincided with high leucine incorporation. Depending on the stage of the cycle JH III exhibited stimulatory or inhibitory effects, or caused no change in RNA synthesis. JH III stimulated RNA synthesis in glands from stages in the reproductive cycle with high endogenous RNA synthesis. The pattern of protein synthesis was not affected by JH III. Ecdysterone lowered RNA synthesis in the LCG and induced secretory activity in LCGs isolated at stages far removed from ovulation. JH III reduced the inhibitory effect of ecdysterone on RNA synthesis and inhibited the ecdysterone-induced secretory activity. The presence of ovarioles isolated at 62 hr of the reproductive cycle (onset of in vivo LCG secretory activity) in the incubation medium inhibited RNA synthesis in 32 hr LCG but induced secretory activity. We suggest that changes in synthetic and secretory activities are coordinated with ovulation and ootheca formation through the interactions between JH III and ovarioles, and ecdysterone may also play a role.

Animals↗
PubMed1976

Double-stranded RNA in chromatin transcripts formed by exogenous RNA polymerase.

RNA transcribed in vitro at low ionic strength, from either rat liver chromatin or DNA, contains a significant amount of structure resistant to RNase in high salt buffer. This is observed with rat liver (form B polymerase) as well as with Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate: RNA nucleotidyltransferase; EC 2.7.7.6). Treatment with RNases specific for either double-stranded or hybrid RNA indicates that resistance to RNase is due to the presence of double-stranded RNA sequences. Denaturation kinetics in the presence or absence of RNase suggest that these sequences are formed by intramolecular base pairing. Their mean length is about 20 to 30 nucleotides, but 15-20% are more than 100 nucleotides long. They contain 60-65% G-C base pairs. The proportion of double-stranded segments is higher in chromatin transcripts than in DNA-templated RNA, and is higher with homologous RNA polymerase than with the bacterial enzyme. On the other hand, chromatin endogenous RNA polymerase, which is unable to initiate transcription, does not synthesize double-stranded RNA. The problem of the location of these sequences is discussed; preliminary results suggest that the 5' end of the RNA transcripts could be enriched in complementary sequences.

Animals↗
PubMed2000

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

5' Untranslated Regions↗