The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells

Jackson, Dean A.; Pombo, Ana; Iborra, Francisco J.

doi:10.1096/fasebj.14.2.242

Cited by 144 publications

(103 citation statements)

References 102 publications

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“…We can imagine a range of possibilities, from each site containing one unit (with 6 -17 engaged polymerases) to 6 -17 units (each with one engaged enzyme). The latter is consistent with our previous results (Cook, 1999;2002), and with observations that few eukaryotic units are associated with more than one engaged polymerase at any moment (reviewed by Jackson et al, 2000). Moreover, microarray analyses reveal only 73 of the several thousand active open reading frames in yeast are typically associated with one or more engaged polymerases (Bon et al, 2006b).…”

Section: Number Of Active Polymerases Per Sitesupporting

confidence: 92%

A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value

Faro-Trindade

Cook

2006

MBoC

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Although we have detailed information on the alterations occurring in steady-state levels of all cellular mRNAs during differentiation, we still know little about more global changes. Therefore, we investigated the numbers of molecules of RNA polymerase II that are active-and the way those molecules are organized-as two mouse cells (aneuploid F9 teratocarcinoma, and euploid and totipotent embryonic stem cells) differentiate into parietal endoderm. Quantitative immunoblotting shows the number of active molecules roughly halves. Transcription sites (detected by light and electron microscopy after allowing engaged polymerases to extend nascent transcripts in bromouridine-triphosphate) are uniformly distributed throughout the nucleoplasm. The numbers of such sites fall during differentiation as nuclei become smaller, but site density and diameter remain roughly constant. Similar site densities and diameters are found in salamander (amphibian) cells with 11-fold larger genomes, and in aneuploid HeLa cells. We conclude that active polymerases and their nascent transcripts are concentrated in a limited number of discrete nucleoplasmic sites or factories, and we speculate that the organization of transcription is conserved during both differentiation and evolution to a high C value. INTRODUCTIONDifferentiation involves the repression of some genes and the activation of others. For example, during erythropoiesis nuclei become smaller and heterochromatic as many genes are silenced; concurrently, globin genes become active by associating with specific nuclear sites known as "factories" or "active chromatin hubs" (Cook, 1999(Cook, , 2002de Laat and Grosveld, 2003;Osborne et al., 2004). Although microarrays provide detailed information on changes in steady-state mRNA levels during differentiation (Kelly and Rizzino, 2000;Harris and Childs, 2002;Tanaka et al., 2002;Sperger et al., 2003), there is little information on how primary rates of transcription change. For example, we do not know by how much global rates vary as any mammalian cell differentiates. Eukaryotic genomes also differ in size more than 200,000-fold in a way unrelated to organismal complexity; there is no consensus as to the basis of this C-value enigma (Hartl, 2000;Gregory, 2001), or on how transcription might be organized in such differently sized genomes.Against this background, we investigated the changes that occur in the rates and organization of transcription as two mouse cells differentiate. Mouse F9 teratocarcinoma cells are a well-studied aneuploid line that can be induced to differentiate into parietal endoderm, which is characterized by flattened cells that secrete tissue-type plasminogen activator, laminin, and type IV collagen (Strickland and Mahdavi, 1978;Strickland et al., 1980;Hogan et al., 1983). ESF/48 -1 cells are totipotent embryonic stem (ES) cells with a normal karyotype; when transferred into host blastocysts, they contribute to all tissues (including germ lines) of the resulting chimeras (Brook and Gardner, 1997), and they can be induc...

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Section: Number Of Active Polymerases Per Sitesupporting

confidence: 92%

A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value

Faro-Trindade

Cook

2006

MBoC

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“…S6 and Materials and Methods). Moreover, the density of RNAP II molecules observed was found to be 37.2 ± 4.1 molecules per μm 3 (n = 12,482 molecules in eight cells), which translates to a total of 80,200 ± 8,800 RNAP II molecules in the entire nucleus (Materials and Methods), in good agreement with values reported in previous studies (13,15,37). Therefore, we conclude that we indeed exhaustively imaged most of the labeled RNAP II molecules in the optical section illuminated.…”

Section: One-to-one Labeling and Superresolution Imaging Of Rnap II Msupporting

confidence: 91%

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

Zhao

Roy

Gebhardt

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

131

105

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Significance We developed an optical imaging technique that combines reflected light-sheet illumination with superresolution microscopy, allowing us to image inside mammalian nuclei at subdiffraction-limit resolution and to count biomolecules with single-copy accuracy. Applying this technique to probe the spatial organization of RNA polymerase II-mediated transcription, we found that the majority of the transcription foci consist of only one RNAP II molecule, contrary to previous proposals. By quantifying the global extent of clustering across RNAP II molecules in the nucleus, we provide clear and convincing answers to the controversy surrounding the prevalent existence of “transcription factories.” Moreover, our work presents imaging and analysis tools for the quantitative characterization of nuclear structures, which could be generally applied to probe many other mammalian systems.

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“…Of all the RNA transcribed in human nuclei, only about 5% enters the cytoplasm 72 . Quality control mechanisms, such as nonsense-mediated decay, dispose of incompletely or improperly processed messages encoding flawed proteins 73 .…”

Section: Rna-directed Rewriting Of Rnamentioning

confidence: 99%

The four Rs of RNA-directed evolution

Herbert

2003

Nat Genet

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The read-out of genetic information in eukaryotes is not only more complex than previously realized, but also more dynamic. Much of the information is 'soft-wired' , with extensive RNA processing necessary to generate phenotypes 10 . A subset of the RNA, referred to here as coRNA ( Fig. 1), coordinates the read-out of genetic information and reconciles regulatory inputs. For each cell type, the sum of these interactions defines a distinct RNA profile or ribotype 10 . RNA-based processes also facilitate replication of ribotypes in subsequent generations. The flexibility of these programs allows soft-wired organisms to evolve in a more rapid and dynamic way than 'hard-wired' organisms constrained by DNA mutation 10 . Together, the actions of reading, 'riting, 'rithmetic and replication constitute the four Rs of RNA-directed evolution. Here, we describe roles for coRNAs and SATs (Fig. 2) in these events. RNA-directed RNA readoutRNA coregulates the sequence-specific read-out of genetic information from RNA by targeting the evolutionarily conserved machinery of RNA interference (RNAi) 11 and translational repression. RNAi leading to post-transcriptional gene silencing (PTGS) is induced by doublestranded RNA (dsRNA) arising from infection by dsRNA viruses, senseantisense transcription pairs, transcription of inverted repeats and delivery of dsRNAs manufactured in vitro 12 . Both endogenous and exogenous dsRNAs are processed cytoplasmically by RNase III dicer paralogs into small interfering RNAs (siRNAs) that are 21-25 bases long 12,13 . These siRNAs are incorporated into an RNA-induced silencing complex (RISC) that targets any RNA with a sequence complementary to the siRNA 12,14 . The target RNA is cleaved by RISC, which has endonucleolytic and presumed exonucleolytic activity 12,15 . Each cycle results in the destruction of the target RNA and the regeneration of an active RISC 16,17 .In Neurospora crassa 18,19 , plants 20 , Schizosaccharomyces pombe 21 and Caenorhabditis elegans 22 , but not in flies, humans or mouse oocyctes 18,19,23,24 , PTGS can also be initiated by an RNA-dependent RNA polymerase (RdRP) that synthesizes complementary RNA from highly expressed transcripts to generate dsRNA. This process can spread from the 3´ to the 5´ end of the mRNA, leading to progressive silencing of a gene and other mRNAs with exons in common (transitive RNAi) 18,22 . From the evolutionary viewpoint, transitive RNAi favors the generation of new phenotypes, as related genes in species with polyploid genomes will escape cosuppression by sequence divergence, resulting in altered function or expression.The number of dicer paralogs also differs between species. Arabidopsis, which has at least four dicer family members, seems to use different enzymes to defend against viruses and to process endogenously produced dsRNA 13,25 . In humans, mice and worms, there is only one dicer ortholog, suggesting that any pathway-specific functions are guided by ancillary regulatory proteins associated with RISC. These ancillary proteins include me...

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The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells

Cited by 144 publications

References 102 publications

A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value

A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

The four Rs of RNA-directed evolution

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