scholarly journals Single-molecule imaging suggests compact and spliceosome dependent organization of long introns

2021 ◽  
Author(s):  
Srivathsan Adivarahan ◽  
A.M.S.Kalhara Abeykoon ◽  
Daniel Zenklusen
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Splice Site ◽  
Spatial Organization ◽  
Spliceosome Assembly ◽  
U2 Snrnp ◽  
Rna In Situ Hybridization ◽  
Intron Removal ◽  
In Cells

Intron removal from pre-mRNAs is a critical step in the processing of RNA polymerase II transcripts, required to create translation competent mRNAs. In humans, introns account for large portions of the pre-mRNA, with intronic sequences representing about 95% of most pre-mRNA. Intron length varies considerably; introns can be as short as a few to hundreds of thousands of nucleotides in length. How nascent long intronic RNA is organized during transcription to facilitate the communication between 5′ and 3′ splice-sites required for spliceosome assembly however is still poorly understood. Here, we use single-molecule fluorescent RNA in situ hybridization (smFISH) to investigate the spatial organization of co- and post-transcriptional long introns in cells. Using two long introns within the POLA1 pre-mRNA as a model, we show that introns are packaged into compact assemblies, and when fully transcribed, are organized in a looped conformation with their ends in proximity. This organization is observed for nascent and nucleoplasmic pre-mRNAs and requires spliceosome assembly, as disruption of U2 snRNP binding results in introns with separated 5′ and 3′ ends. Moreover, interrogating the spatial organization of partially transcribed co-transcriptional POLA1 intron 35 indicates that the 5′ splice site is maintained proximal to the 3′ splice site during transcription, supporting a model that 5′ splice site tethering to the elongating polymerase might contribute to spliceosome assembly at long introns. Together, our study reveals details of intron and pre-mRNA organization in cells and provides a tool to investigate mechanisms of splicing for long introns.

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 TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation

2021 ◽  
Vol 23 (1) ◽  
pp. 107
Author(s):  
Matthias Vanneste ◽  
Marie Mulier ◽  
Ana Cristina Nogueira Freitas ◽  
Nele Van Ranst ◽  
Axelle Kerstens ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Single Molecule ◽  
Drg Neurons ◽  
Functional Responses ◽  
Internal Organs ◽  
Rna In Situ Hybridization ◽  
Deficient Mice ◽  
Heat Hyperalgesia

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

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.

scholarly journals Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations

2020 ◽  
Author(s):  
Leah Bury ◽  
Brittania Moodie ◽  
Liliana S. McKay ◽  
Karen H. Miga ◽  
Iain M. Cheeseman
Keyword(s):  
Cell Division ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Alpha Satellite ◽  
Satellite Rna ◽  
Chromatin Dynamics ◽  
Rna Transcripts ◽  
Centromere Proteins ◽  
Non Coding Rnas

AbstractCentromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alpha-satellite transcript levels increase substantially when the nucleolus is disrupted. Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

scholarly journals Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Leah Bury ◽  
Brittania Moodie ◽  
Jimmy Ly ◽  
Liliana S McKay ◽  
Karen HH Miga ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Single Molecule ◽  
Alpha Satellite ◽  
Satellite Rna ◽  
Single Molecule Fluorescence ◽  
Chromatin Dynamics ◽  
Rna Transcripts ◽  
Non Coding Rnas

Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere–nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

scholarly journals Spatial organization of the somatosensory cortex revealed by cyclic smFISH

2018 ◽  
Author(s):  
Simone Codeluppi ◽  
Lars E. Borm ◽  
Amit Zeisel ◽  
Gioele La Manno ◽  
Josina A. van Lunteren ◽  
...  
Keyword(s):  
Single Cell ◽  
Somatosensory Cortex ◽  
Single Molecule ◽  
Spatial Organization ◽  
Spatial Information ◽  
Cell Types ◽  
Cellular Organization ◽  
New Methods ◽  
The Brain

The global efforts towards the creation of a molecular census of the brain using single-cell transcriptomics is generating a large catalog of molecularly defined cell types lacking spatial information. Thus, new methods are needed to map a large number of cell-specific markers simultaneously on large tissue areas. Here, we developed a cyclic single molecule fluorescence in situ hybridization methodology and defined the cellular organization of the somatosensory cortex using markers identified by single-cell transcriptomics.

scholarly journals Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly

2019 ◽  
Vol 47 (20) ◽  
pp. 10842-10851 ◽  
Author(s):  
David H Beier ◽  
Tucker J Carrocci ◽  
Clarisse van der Feltz ◽  
U Sandy Tretbar ◽  
Joshua C Paulson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Conformational Dynamics ◽  
Spliceosome Assembly ◽  
Single Molecule Fret ◽  
Dead Box ◽  
U2 Snrnp ◽  
The Dead

Abstract The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

scholarly journals Characterization of a U2AF-Independent Commitment Complex (E′) in the Mammalian Spliceosome Assembly Pathway

2005 ◽  
Vol 25 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Oliver A. Kent ◽  
Dustin B. Ritchie ◽  
Andrew M. MacMillan
Keyword(s):  
Splice Site ◽  
Early Recognition ◽  
Close Association ◽  
Nuclear Extract ◽  
Sr Protein ◽  
Spliceosome Assembly ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Auxiliary Factor

ABSTRACT Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5′ splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3′ splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E′ which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E′ complex is characterized by a U1 snRNA-5′ splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5′ splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.

scholarly journals Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia

2021 ◽  
Vol 23 (2) ◽  
pp. 184-197 ◽  
Author(s):  
Cindrilla Chumduri ◽  
Rajendra Kumar Gurumurthy ◽  
Hilmar Berger ◽  
Oliver Dietrich ◽  
Naveen Kumar ◽  
...  
Keyword(s):  
Transition Zone ◽  
Single Molecule ◽  
Spatial Organization ◽  
Lineage Tracing ◽  
Stromal Compartment ◽  
Transition Zones ◽  
Wnt Inhibitor ◽  
Stem Cell Populations ◽  
In Situ Hybridizations

AbstractThe transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

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