Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy

ACS Nano ◽  
2016 ◽  
Vol 10 (2) ◽  
pp. 2447-2454 ◽  
Author(s):  
Xuanze Chen ◽  
Mian Wei ◽  
M. Mocarlo Zheng ◽  
Jiaxi Zhao ◽  
Huiwen Hao ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Live Cell ◽  
Cell Nuclei

scholarly journals Correction to Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy

ACS Nano ◽  
2016 ◽  
Vol 10 (4) ◽  
pp. 4882-4882 ◽  
Author(s):  
Xuanze Chen ◽  
Mian Wei ◽  
M. Mocarlo Zheng ◽  
Jiaxi Zhao ◽  
Huiwen Hao ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Live Cell ◽  
Cell Nuclei

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Transcription organizes euchromatin similar to an active microemulsion

2017 ◽  
Author(s):  
Lennart Hilbert ◽  
Yuko Sato ◽  
Hiroshi Kimura ◽  
Frank Jülicher ◽  
Alf Honigmann ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Super Resolution ◽  
Live Cell ◽  
Live Cell Microscopy ◽  
Cell Microscopy

Chromatin is organized into heterochromatin, which is transcriptionally inactive, and euchromatin, which can switch between transcriptionally active and inactive states. This switch in euchromatin activity is accompanied by changes in its spatial distribution. How euchromatin rearrangements are established is unknown. Here we use super-resolution and live-cell microscopy to show that transcriptionally inactive euchromatin moves away from transcriptionally active euchromatin. This movement is driven by the formation of RNA-enriched microenvironments that exclude inactive euchromatin. Using theory, we show that the segregation into RNA-enriched microenvironments and euchromatin domains can be considered an active microemulsion. The tethering of transcripts to chromatin via RNA polymerase II forms effective amphiphiles that intersperse the two segregated phases. Taken together with previous experiments, our data suggest that chromatin is organized in the following way: heterochromatin segregates from euchromatin by phase separation, while transcription organizes euchromatin similar to an active microemulsion.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2020 ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell ◽  
Highly Efficient

The carboxyl-terminal domain of RNA polymerase II is dynamically phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to unphosphorylated and phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as clusters of Serine 5 phosphorylated RNAP2 that form around active genes and are separated in both space and time from nascent mRNA synthesis. Computational models fit to our data determine that 5 to 40 RNAP2 cluster around the promoter of a gene during typical transcriptional bursts. Nearly all RNAP2 either arrive with Serine 5 phosphorylation or acquire the modification within a minute. Transcription from the cluster appears to be highly efficient, with nearly half of the clustered RNAP2 ultimately escaping the promoter in a minute or so to elongate a full-length mRNA in approximately five minutes. The highly dynamic and spatially organized concentrations of RNAP2 we observe support the notion of highly efficient transcription clusters that form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

Transcription boundaries of U1 small nuclear RNA

1985 ◽  
Vol 5 (9) ◽  
pp. 2332-2340
Author(s):  
G R Kunkel ◽  
T Pederson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Cell Nuclei ◽  
Sm Proteins ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Rna Biosynthesis ◽  
Labeled Rna ◽  
Alpha Amanitin

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

scholarly journals Transcription boundaries of U1 small nuclear RNA.

1985 ◽  
Vol 5 (9) ◽  
pp. 2332-2340 ◽  
Author(s):  
G R Kunkel ◽  
T Pederson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Cell Nuclei ◽  
Sm Proteins ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Rna Biosynthesis ◽  
Labeled Rna ◽  
Alpha Amanitin

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

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