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Dynamic organization of transcription compartments is dependent on functional nuclear architecture.

ABSTRACT: Transcription in higher organisms requires spatiotemporal coordination of transcription machinery and the transcription factors at promoter sites. Toward this, recent evidence suggests that both static compartmentalization and dynamic self-organization of transcriptional apparatus are in effect at sites of transcription. Although the dynamics of transcription machinery is essential to genome regulation, the principles underlying this organization and its functional coupling to nuclear architecture is unclear. In a recent study we revealed that Uridine-5'-triphosphate (UTP) uptake in living cells labeled transcription-related compartments. In this article, we quantitatively establish multicolor labeling strategies for UTP-enriched transcription compartments (TCs) and probe their dynamic organization. UTP-enriched TCs were found to be in two distinct fractions: one colocalized with phosphorylated RNA pol II and the other as nascent aggregates. The fraction colocalized with the phosphorylated RNA pol II decreased with the inhibition of transcription initiation or elongation. Fluorescence anisotropy imaging and photobleaching experiments suggest that TCs are functional aggregates of nascent transcripts that are assembled in a transcription-dependent manner. Fluorescence correlation spectroscopy analysis revealed the relative fraction and sizes of fluorescent UTP-labeled transcripts in the nucleoplasm. Time-lapse imaging experiments of TCs exhibited pause and a mobile nature of these compartments within interchromosome territories. Perturbation of either nucleoskeletal protein or the cytoskeleton resulted in reduced active mobility of TCs, whereas inhibitors of transcription enhanced the mobile fraction of TCs. Further, high temporal resolution imaging showed evidence of stepping dynamics of TCs regulated by nucleoskeleton and chromatin modifications. Taken together, our experiments suggest the transient compartmentalization of UTP-enriched aggregates and their dynamic reorganization in a transcription-dependent manner. These results may have important implications for understanding spatiotemporal control of eukaryotic transcription.

SUBMITTER: Maharana S

PROVIDER: S-EPMC3433616 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Dynamic organization of transcription compartments is dependent on functional nuclear architecture.

Maharana Shovamayee S Sharma Divya D Shi Xianke X Shivashankar G V GV

Biophysical journal 20120901 5

Transcription in higher organisms requires spatiotemporal coordination of transcription machinery and the transcription factors at promoter sites. Toward this, recent evidence suggests that both static compartmentalization and dynamic self-organization of transcriptional apparatus are in effect at sites of transcription. Although the dynamics of transcription machinery is essential to genome regulation, the principles underlying this organization and its functional coupling to nuclear architectu ...[more]

PMID: 23009834

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Project description:BACKGROUND:The magnitude of intronic and intergenic DNA can vary substantially both within and among evolutionary lineages; however, the forces responsible for this disparity in genome compactness are conjectural. One explanation, termed the mutational-burden hypothesis, posits that genome compactness is primarily driven by two nonadaptive processes: mutation and random genetic drift - the effects of which can be discerned by measuring the nucleotide diversity at silent sites (pisilent), defined as noncoding sites and the synonymous sites of protein-coding regions. The mutational-burden hypothesis holds that pisilent is negatively correlated to genome compactness. We used the model organism Chlamydomonas reinhardtii, which has a streamlined, coding-dense mitochondrial genome and an noncompact, intron-rich nuclear genome, to investigate the mutational-burden hypothesis. For measuring pisilent we sequenced the complete mitochondrial genome and portions of 7 nuclear genes from 7 geographical isolates of C. reinhardtii. RESULTS:We found significantly more nucleotide diversity in the nuclear compartment of C. reinhardtii than in the mitochondrial compartment: net values of pisilent for the nuclear and mitochondrial genomes were 32 x 10-3 and 8.5 x 10-3, respectively; and when insertions and deletions (indels) are factored in, these values become 49 x 10-3 for the nuclear DNA and 11 x 10-3 for the mitochondrial DNA (mtDNA). Furthermore, our investigations of C. reinhardtii revealed 4 previously undiscovered mitochondrial introns, one of which contains a fragment of the large-subunit (LSU) rRNA gene and another of which is found in a region of the LSU-rRNA gene not previously reported (for any taxon) to contain introns. CONCLUSION:At first glance our results are in opposition to the mutational-burden hypothesis: pisilent was approximately 4 times greater in the nuclear compartment of C. reinhardtii relative to the mitochondrial compartment. However, when we consider the encumbrance of noncoding DNA in each of these C. reinhardtii compartments, we conclude that introns in the mtDNA impose a greater burden than those in the nuclear DNA and suggest that the same may be true for the intergenic regions. Overall, we cannot reject the mutational-burden hypothesis and feel that more data on nucleotide diversity from green algae and other protists are needed.

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Dynamic organization of transcription compartments is dependent on functional nuclear architecture.

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Dynamic organization of transcription compartments is dependent on functional nuclear architecture.

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