Landscape of transcription in human cells (original) (raw)
Related papers
The EMBO Journal, 1999
As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that ∼10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only ∼2000 sites (diameter ∼40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures.
2011
Background: In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates. Results: Using microarrays comprising oligonucleotide probes that represent either proteincoding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions. Conclusion: Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.
Cold Spring Harbor Perspectives in Biology, 2010
Investigations into the organization of transcription have their origins in cell biology. Early studies characterized nascent transcription in relation to discernable nuclear structures and components. Advances in light microscopy, immunofluorescence, and in situ hybridization helped to begin the difficult task of naming the countless individual players and components of transcription and placing them in context. With the completion of mammalian genome sequences, the seemingly boundless task of understanding transcription of the genome became finite and began a new period of rapid advance. Here we focus on the organization of transcription in mammals drawing upon information from lower organisms where necessary. The emerging picture is one of a highly organized nucleus with specific conformations of the genome adapted for tissue-specific programs of transcription and gene expression.
BMC Genomics, 2007
In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates.
Sub-nuclear fractionation II. Intranuclear compartmentation of transcription in vivo and in vitro
Experimental Cell Research, 1974
DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper . Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to a-amanitin, exogenous native and denatured DNA, thermal denaturation at 45, Mg2+ and Mn2 ions, high ionic strength and by the binding of '*C-methyl-y-amanitin. RNA polymerase B (a-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of cc-amanitin subnuclear fractions that had been pre-incubated at 45°C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.
Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei
Experimental Cell Research, 2011
The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Introductory Chapter: Transcriptome Analysis
Transcriptome Analysis [Working Title]
The central dogma of molecular biology describes the flow of genetic information from genes to functions of the cells and organisms. This comprises a two-step process: first, DNA, the permanent, heritable, genetic information repository, is transcribed by the RNA polymerase enzymes into RNA, a short-lasting information carrier; second, a subset of RNA, the messenger RNAs, mRNAs, are translated into protein. The transcriptome, then, is the complete set of all RNA molecules in a cell, a population of cells or in an organism. Importantly, not all RNAs are translated into proteins, some serve a structural function, for example, rRNAs in the assembly of ribosomes, others are transporters, e.g., tRNAs, yet others serve regulatory functions, for example, the siRNAs, short interfering RNA, or lncRNAs, long non-coding RNAs; these are not translated into proteins [1]. However, these non-coding RNAs can and often do play roles in human diseases such as cancer, cardiovascular, and neurological disorders. While transcriptomics is most commonly applied to the mRNAs, the coding transcripts, transcriptomics also provides important data regarding content of the cell noncoding RNAs, including rRNA, tRNA, lncRNA, siRNA, and others. Specific approaches address the analysis of splice variant of the same gene in different tissues.