In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA

Koberna, Karel; Staněk, David; Malínský, J; Čtrnáctá, Vlasta; Cermanová, Štěpánka; Novotná, Jana; Kopský, V.; Raška, Ivan

doi:10.1078/0065-1281-00535

Cited by 6 publications

(11 citation statements)

References 8 publications

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“…We faced, however, a major technical problem in detection of labeled RNA as far as the consecutive pulses of different modified nucleotides were concerned (see below). While biotin-labeled RNA can be detected regardless of the fixation method used, BrU-labeled RNA fails to be detected, without additional treatment, in nucleoli after formaldehyde fixation (Wansink et al 1993;Koberna et al 1999Koberna et al , 2000. Nucleolar BrU-labeled RNA, BrU representing the most convenient marker of newly synthesized RNA in general, requires methanol/acetone fixation for detection (Koberna et al 1999(Koberna et al , 2000.…”

Section: Fibrillarin Domains Are Not Affected During Hypotonic Treatmentmentioning

confidence: 97%

“…While biotin-labeled RNA can be detected regardless of the fixation method used, BrU-labeled RNA fails to be detected, without additional treatment, in nucleoli after formaldehyde fixation (Wansink et al 1993;Koberna et al 1999Koberna et al , 2000. Nucleolar BrU-labeled RNA, BrU representing the most convenient marker of newly synthesized RNA in general, requires methanol/acetone fixation for detection (Koberna et al 1999(Koberna et al , 2000. This latter approach represents fixation by precipitation rather than true chemical fixation, and therefore, using light microscopy resolution, we had to demonstrate that compatible results can be obtained after both formaldehyde and methanol/acetone fixation.…”

Section: Fibrillarin Domains Are Not Affected During Hypotonic Treatmentmentioning

confidence: 98%

“…This protocol, in contrast to formaldehyde fixation, allows light microscopy detection of nucleolar BrU-labeled RNA in non-sectioned cells (Koberna et al 2000). After washing with PBS, the cells expressing GFP-fibrillarin were incubated with a mouse anti-BrdU antibody (Exbio, Prague, Czech Republic) and then with a secondary anti-mouse antibody conjugated with Cy3 (Jackson ImmunoResearch Laboratories, WestGrove, Pa.).…”

Section: Indirect Immunofluorescencementioning

confidence: 99%

“…In order to achieve this aim, we utilized a recently developed method of hypotonic shift for RNA labeling with modified nucleotides in living cells (Koberna et al 1999) and the detection of newly synthesized nucleolar RNA without any drug treatment (Koberna et al 2000). In contrast to microinjection (Cmarko et al 1999(Cmarko et al , 2000, this method allows the labeling of a large number of cultured cells at the same time.…”

Section: Introductionmentioning

confidence: 99%

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Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus

et al. 2001

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The precise location of ribosomal RNA (rRNA) synthesis within the nucleolus is the subject of recent controversy; some investigators have detected nascent RNA in the dense fibrillar components (DFCs) while others have localized transcription to the fibrillar centers (FCs). We endeavored to resolve this controversy by applying a new technique for non-isotopic labeling of RNA and examined the synthesis and movement of non-isotopically labeled rRNA within the nucleolus. We found that rRNA is synthesized only in a restricted area of DFCs, also involving the boundary region with FCs. We traced a movement of RNA from transcription sites through DFCs to granular components. Our results indicate functional compartmentalization of DFCs with respect to the synthesis and processing of precursor rRNA. In situ mapping of the 5' leader sequence of the 5' external transcribed spacer together with transcription labeling indicated that transcription and the first steps in processing of precursor rRNA are spatially separated. Surprisingly, the results also pointed to a partially extended conformation of newly synthesized precursor rRNA transcripts.

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Section: Fibrillarin Domains Are Not Affected During Hypotonic Treatmentmentioning

confidence: 97%

Section: Fibrillarin Domains Are Not Affected During Hypotonic Treatmentmentioning

confidence: 98%

Section: Indirect Immunofluorescencementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus

et al. 2001

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“…) in glycerol buffer [25 % (w/v) glycerol, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM PMSF, 20 mM Tris/HCl (pH 7.4)] and exposed to transcription buffer [25 % (w/v) glycerol, 100 mM KCl, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM each ATP, CTP and GTP, 0.2 mM BrUTP (Sigma), 1 mM PMSF, 5 U RNAsin ml 21 (Promega), 50 mM Tris/HCl (pH 7.4)] for 30 min at 37 u C as described previously (Koberna et al, 2000). After incubation, the coverslips were fixed in cold 1 : 1 methanol : acetone mix at 220 u C for 4 min, dried and stained with anti-BrUTP monoclonal antibody (clone BU-33; Sigma) and Hoechst-33342 DNA dye as described previously (Bardina et al, 2009 The control cells displayed a moderate granular staining in the nucleoplasm that corresponded to RNA pol II-and pol III-dependent synthesis, which could not be discriminated by this method.…”

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confidence: 99%

Variability in inhibition of host RNA synthesis by entero- and cardioviruses

Krupina

Sheval

Lidsky

2010

Journal of General Virology

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cAMP‐dependent reorganization of the Cajal bodies and splicing machinery in cultured Schwann cells

Fernandez

Pena

Navascués

et al. 2002

Glia

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It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression.

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In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA

Cited by 6 publications

References 8 publications

Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus

Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus

Variability in inhibition of host RNA synthesis by entero- and cardioviruses

cAMP‐dependent reorganization of the Cajal bodies and splicing machinery in cultured Schwann cells

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