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PubMed2026

CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.

Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.

CRISPR↗
PubMed1984

Cyclosporin A is a differential inhibitor of eukaryotic RNA polymerases.

In order to investigate the molecular mechanism of biological action of the drug cyclosporin A (CsA), we have studied its effect on eukaryotic RNA polymerase activities in two systems: (a) in vitro transcription; (b) injection into frog oocytes. The electron microscopic transcription R-loop method [1] has been used to assay for specific transcription of an immunoglobulin gene clone in HeLa cell lysates. In this system, CsA inhibits efficiently the transcription of immunoglobulin genes. Dilution experiments show that RNA polymerase II activity is inhibited by nanomolar concentrations (i.e. amounts comparable to the ones needed for the inhibition of lymphocyte stimulation), whereas polymerase III is inhibited at micromolar concentrations. In vitro transcription with E. coli RNA polymerase is not inhibited. Parallel experiments with the mushroom toxin alpha-amanitin have shown that the eukaryotic RNA polymerase II displays similar sensitivity towards the two cyclic peptides CsA and alpha-amanitin in vitro. When CsA is injected into frog oocytes, together with a cloned Xenopus laevis U1 small nuclear RNA gene, transcription of this gene by endogenous RNA polymerase II is inhibited by 60%. RNA polymerase III and RNA polymerase I transcription is not inhibited in the oocyte. Various possibilities for the selective action of CsA on stimulated lymphocytes are discussed.

Amanitins↗
PubMed2002

RNA interference (RNAi) inhibits growth of Plasmodium falciparum.

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P<0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P<0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets.

Animals↗
PubMed1976

Fidelity of chromatin transcription in vitro.

Chromatin and DNA from Schneider's Drosophila melanogaster cell line 2 were transcribed in vitro with Escherichia coli RNA polymerase. Using mercurated UTP as precursor, the newly synthesized RNA could be separated from DNA and endogenous RNA by affinity chromatography on sulfhydryl-Sepharose 6B. Characterization of the transcription products with complementary DNA (cDNA) made from polyadenylated nuclear RNA and with fractionated cDNA probe demonstrated a fair quantitative fidelity in the in vitro transcript from chromatin which was not evident when DNA was transcribed. However, as shown by hybridization to total nuclear RNA, E. coli RNA polymerase transcribed both DNA strands from chromatin in vitro. We conclude that E. coli polymerase is able to distinguish sections of chromatin at which rapid synthesis of RNA occurs in the cell.

Cell Line↗
PubMed1976

In vitro transcription of chromatin in the presence of a mercurated nucleotide.

Mercurated uridine triphosphate has been used to label transcripts of chicken reticulocyte chromatin made with Escherichia coli RNA polymerase. The mercury-labeled RNA product can be completely separated from endogenous RNA sequences in the chromatin by passage through a sulfhydryl Sepharose column. Globin cDNA hybridization to the transcript shows that only 2.6 x 10-5 of the transcript is globin RNA. In contrast to this result, erythrocte chromatin transcript contains less than one tenth as many globin RNA sequences.

Animals↗
PubMed1973

RNA-DNA covalent bonds between the RNA primers and the DNA products formed by RNA tumor virus DNA polymerase.

Initiation of DNA synthesis by endogenous RNA primer molecules was studied with three different RNA tumor viruses. The influence of the method of virus disruption on the observed RNA-DNA bonds was ascertained. Ether disrupted virions of both murine leukemia virus (MuLV) and the B77 strain of avian sarcoma virus (B77 virus) have rC-dC and rA-dA covalent linkages between RNA primers and newly synthesized DNA. None of the 14 other possible bonds were formed. Ether-disrupted virions of avian myeloblastosis virus (AMV) have rU-dC and rA-dA linkages. In contrast, work reported herein and from other laboratories shows that Nonidet P-40 (NP-40)-disrupted virions of all three viruses have only the rA-dA junction. Studies with virus particles which were first disrupted with ether and then treated with NP-40 indicated that the detergent treatment disallowed the formation of the ribopyrimidine-dC internucleotide bond. The same transfers are found with AMV in the presence or absence of actinomycin D, where only single-stranded DNA is formed. This finding is consistent with the notion that virtually all of the significant primers have been recognized. In contrast to mature virions, transfer experiments with ether-disrupted early harvest (5 min) MuLV showed only the rC-dC bond; the rA-dA bond was absent. The short-time harvest contains a significantly higher proportion of infectious virions than 24-h harvests. Also, since the RNA from early harvest virus is appreciably more homogenous than the RNA of mature MuLV, it is concluded that the ribopyrimidine-dC linkage is the more significant initiation event from a biochemical standpoint.

Alpharetrovirus↗
PubMed1995

The activity of transcription factor PBP, which binds to the proximal sequence element of mammalian U6 genes, is regulated during differentiation of F9 cells.

Mouse F9 embryonic carcinoma (EC) cells differentiate in culture to parietal endoderm (PE) cells upon induction with retinoic acid and cyclic AMP. In the course of this process, the expression of polymerase III transcripts, e.g., 5S rRNA and U6 small nuclear RNA, is dramatically reduced. This reduction of endogenous RNA content is accompanied by a loss of transcriptional capacity in cell extracts from PE cells. Partial purification of such extracts reveals that the DNA-binding activity of transcription factor PBP, binding specifically to the proximal sequence element (PSE) sequence of vertebrate U6 genes, is significantly reduced. This finding is corroborated by a loss in the transcriptional activity of this factor in reconstitution assays with partially purified polymerase III transcription components. In contrast, the activity of TFIIIA and TFIIIB and the amount of free TATA-binding protein remain unchanged during the differentiation process analyzed here. These data show for the first time that the PSE-binding protein PBP is essentially involved in the differential regulation of polymerase III genes governed by external promoters.

Animals↗
PubMed1980

Ribonucleic acid synthesis in the myocardium of spontaneously hypertensive rats. Quantification of transcribing ribonucleic acid polymerases.

Cardiac hypertrophy accompanies the progressive rise in blood pressure in spontaneously hypertensive rats. The role of endogenous RNA polymerases in this process was examined in nuclei from isolated cardiac myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Both template-engaged (involved in transcription) and free (loosely attached to endogenous template, transcribing only with exogenous templates) RNA polymerases were increased in spontaneously hypertensive rats. In addition, the ratio of RNA polymerases I/II was lower in the spontaneously hypertensive rats for both functional pools of the enzyme. Endogenous transcribing RNA polymerases were quantified by t.l.c. of RNA-hydrolysis products. Increased numbers of enzyme molecules were present in nuclei from spontaneously hypertensive rats, without appreciable change in the rate of polyribonucleotide-chain elongation. These results could not be explained by differences in the activities of contaminating phosphatases or ribonucleases, nor by changes in endogenous nucleoside pools or recoveries of labelled nucleosides. Enhanced myocardial RNA synthesis in the spontaneously hypertensive rats at the stage of established cardiac hypertrophy is associated with increased numbers of RNA polymerase molecules. This increase may, in turn, reflect altered chromatin structure, resulting in increased polymerase binding and/or chain initiation.

Animals↗
PubMed1979

RNA synthesis in permeable mouse ascites sarcoma cells.

A permeable cell system for studying RNA synthesis was established. Mouse ascites sarcoma cells were made permeable to nucleoside triphosphates and alpha-amanitin by treating with a hypotonic buffer. Separate determinations of endogenous RNA polymerase I, II and III activities in permeable cells were conducted using the different sensitivities of these enzymes to alpha-amanitin. The endogenous activity of RNA polymerase II under optimal conditions was one tenth of total RNA synthetic activity in isolated nuclei, and one third of that in permeable cells. The extremely low ratio of RNA polymerase II activity to total RNA synthetic activity in isolated nuclei was thought to be caused by increase of RNA polymerase I activity and decrease of RNA polymerase II activity. These and other results suggested that RNA synthesis in permeable cells reflects more precisely the in vivo state of RNA synthesis than thatin isolated nuclei. The permeable cell system will provide a useful method for studying the separate activities of RNA polymerases I, II and III in situ.

Amanitins↗
PubMed1978

On the activity of RNA polymerase B in lysates from Ehrlich ascites cells.

Transcription by endogenous RNA polymerase B in lysates of Ehrlich ascites cells was investigated. The enzyme exhibits two salt optima at 0.025 M and at 0.3 M (NH4)2SO4 respectively. Preincubation of the cells with the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole results in an inactivation of the polymerase molecules active under condition of low salt. This indicates two functional states of the enzyme in vivo. Initiations of RNA chains by polymerase B do not occur in vitro as judged by the incorporation of [beta-32P]GTP. Thus the two functional states seem to be both elongating polymerase molecules. Polymerase B does not occur in the lysates in a state ready to initiate on an exogenous template, in contrast to polymerase A and C which do occur in free form. Pretreatment with dichlororibofuranosylbenzimidazole in vivo does not result in an accumulation of free polymerase B.

Animals↗
PubMed1977

Transcription of specific genes in isolated nuclei by exogenous RNA polymerases.

Mouse plasmacytoma (MOPC) 460 cells contain two chromatographic forms of RNA polymerase III (IIIA and IIIB) in addition to the major class I and II RNA polymerases. Nuclei isolated from these cells actively synthesize RNA. Among the discrete transcription products observed are the 5S and 4.5S RNAs and additional low molecular weight RNA species (approximately 5.8S, 6.3S, and 6.6S in size). The 4.5S RNAs appear to be tRNA precursors since they can be converted in vitro to 4S RNAs. Studies with alpha-amanitin have shown that the synthesis of these discrete RNA species, and other uncharacterized transcripts somewhat larger in size, is mediated by an endogenous RNA polymerase III activity(ies). Nuclear RNA synthesis is stimulated by exogenous purified RNA polymerases. Exogenous MOPC class III RNA polymerases stimulate the synthesis of each of the distinct low molecular weight species (including 5S and 4.5S RNAs) about 3-6 fold. The hybridization of nuclear transcripts to purified 5S genes (5S DNA) confirms that exogenous class III RNA polymerases stimulate (approximately 4 fold) the synthesis of ribosomal 5S RNA. The 5S RNA genes in nuclei are transcribed asymmetrically by both the endogenous and the exogenous class III enzymes. Exogenous RNA polymerase III from Xenopus laevis ovaries stimulates 4.5S and 5S RNA synthesis in MOPC nuclei as effectively as do the MOPC class III RNA polymerases. However, exogenous MOPC class I and II RNA polymerases do not stimulate 4.5S and 5S RNA synthesis, suggesting that this effect is specific for the structurally similar class III RNA polymerases.

Amanitins↗
PubMed1979

Comparison of endogenous and exogenous RNA primers of poly(U) polymerase in rat hepatic ribosomes.

The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on Sephadex G-150 and CM-cellulose. Our results demonstrate the presence of several forms of poly(U) polymerase activities, some requiring exogenous RNA and others possessing their own endogenous primer RNA.

Animals↗
PubMed1985

Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei.

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.

Adenosine Triphosphatases↗
PubMed1977

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Murine erythroleukemic cells accumulate cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly; globin RNA synthesis by isolated erythroleukemic cell nuclei was studied. Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP). Mercurated RNA synthesized in vitro was purified free of endogenous RNA by affinity chromatography on sulfhydryl-Sepharose, and analyzed for the presence of newly synthesized globin RNA sequences by molecular hybridization to globin complementary [32P]DNA. The results demonstrate markedly increased synthesis of globin RNA by nuclei isolated from dimethyl sulfoxide-treated cells, even within 5 min of nuclear transcription in vitro. These findings are most consistent with transcriptional activation of the globin genes upon induction of differentiation.

Cell Differentiation↗
PubMed2001

RNA association defines a functionally conserved domain in the nuclear pore protein Nup153.

Traffic between the nucleus and cytoplasm takes place through a macromolecular structure termed the nuclear pore complex. To understand how the vital process of nucleocytoplasmic transport occurs, the contribution of individual pore proteins must be elucidated. One such protein, the nucleoporin Nup153, is localized to the nuclear basket of the pore complex and has been shown to be a central component of the nuclear transport machinery. Perturbation of Nup153 function was demonstrated previously to block the export of several classes of RNA cargo. Moreover, these studies also showed that Nup153 can stably associate with RNA in vitro. In this study, we have mapped a domain within Nup153, encompassing amino acids 250-400 in human Nup153, that is responsible for RNA association. After cloning this region of Xenopus Nup153, we performed a cross-species analysis. Despite variation in sequence conservation between Drosophila, Xenopus, and human, this domain of Nup153 displayed robust RNA binding activity in each case, indicating that this property is a hallmark feature of Nup153 and pointing toward a subset of amino acid residues that are key to conferring this ability. We have further determined that a recombinant fragment of Nup153 can bind directly to RNA and that this fragment can interact with endogenous RNA targets. Our findings identify a functionally conserved domain in Nup153 and suggest a role for RNA binding in Nup153 function at the nuclear pore.

Amino Acid Sequence↗
PubMed1981

Characterization of RNA polymerase products of Nebraska calf diarrhea virus and SA11 rotavirus.

The endogenous RNA polymerase of the calf rotavirus was shown to synthesize single-stranded RNA transcripts of one polarity which were identical in size to the denatured parental double-stranded RNA segments. The transcripts were notable in their absence of polyadenylate sequences. The polymerase activity associated with the cores of calf rotavirus was minimally altered by the reaction, since calf rotavirus cores could be reused after purification, whereas the RNA polymerase products appeared to be released from the virion.

Base Sequence↗
PubMed2001

A framework for the molecular classification of circulating tumor markers.

New molecular biological technologies, especially polymerase chain reaction (PCR) and mass spectroscopy, have expanded the pool of molecular targets for cancer diagnosis, monitoring, and prognosis using plasma or serum as the substrate. In this review, a framework is described following the "life history" of a protein--starting with DNA [endogenous (nuclear or mitochondrial) or exogenous (viral)], followed by RNA [endogenous (cell-based or cell-free) or exogenous (viral)], and culminating in protein (either the native protein or the glycan portion of glycoproteins). Each of these levels provides unique opportunities to achieve specificity for cancer diagnosis.

Biomarkers, Tumor↗
PubMed2004

Prions at the crossroads: the need to identify the active TSE agent.

Structural change in the cellular prion protein, PrPC to a ProteinaseK-resistant beta-sheet-rich insoluble form PrPSC and its accumulation have been considered to be central to the pathogenesis of the prion diseases (TSE). In a recent paper, Deleault et al have shown that specific endogenous RNA molecules can induce in vitro structural conversion of endogenous PrPC to PrPSC. Small highly structured synthetic RNAs can also induce this conversion process. However, recent in vivo results show that PrPSC is not directly involved in the prion pathogenesis. It is possible, however, that nucleic-acid-induced PrPSC associated with the inducer nucleic acid could be the components of the infectious agent.

Animals↗