Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

AbstractXist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA.One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

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Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

Science ◽

10.1126/science.abe7500 ◽

2021 ◽

Vol 372 (6547) ◽

pp. eabe7500

Author(s):

Lisa Rodermund ◽

Heather Coker ◽

Roel Oldenkamp ◽

Guifeng Wei ◽

Joseph Bowness ◽

...

Keyword(s):

Dynamic Properties ◽

Super Resolution ◽

Feedback Mechanism ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Time Resolved ◽

Rna Molecules ◽

Fundamental Parameters ◽

Xist Rna ◽

Key Factor

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.

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X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Development ◽

10.1242/dev.127.19.4137 ◽

2000 ◽

Vol 127 (19) ◽

pp. 4137-4145 ◽

Cited By ~ 4

Author(s):

I. Okamoto ◽

S. Tan ◽

N. Takagi

Keyword(s):

X Chromosome ◽

Ectopic Expression ◽

Blastocyst Stage ◽

X Chromosome Inactivation ◽

X Inactivation ◽

Chromosome Inactivation ◽

Current View ◽

Replication Banding ◽

Xist Rna ◽

Morula Stage

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

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Localized accumulation of Xist RNA in X chromosome inactivation

Open Biology ◽

10.1098/rsob.190213 ◽

2019 ◽

Vol 9 (12) ◽

pp. 190213 ◽

Cited By ~ 2

Author(s):

Neil Brockdorff

Keyword(s):

X Chromosome ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

X Chromosomes ◽

Non Coding Rna ◽

Xist Rna ◽

Analogous Manner ◽

Mammalian Embryos ◽

Non Coding Rnas ◽

In Cis

The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions in cis , affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.

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Impact of Xist RNA on chromatin modifications and transcriptional silencing maintenance at different stages of imprinted X chromosome inactivation in vole Microtus levis

Chromosoma ◽

10.1007/s00412-017-0650-9 ◽

2017 ◽

Vol 127 (1) ◽

pp. 129-139 ◽

Cited By ~ 4

Author(s):

Alexander I. Shevchenko ◽

Elena V. Grigor’eva ◽

Sergey P. Medvedev ◽

Irina S. Zakharova ◽

Elena V. Dementyeva ◽

...

Keyword(s):

X Chromosome ◽

Transcriptional Silencing ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

Chromatin Modifications ◽

Xist Rna ◽

Microtus Levis

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Getting to the center of X-chromosome inactivation: the role of transgenesThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o09-040 ◽

2009 ◽

Vol 87 (5) ◽

pp. 759-766 ◽

Cited By ~ 3

Author(s):

Jakub Minks ◽

Carolyn J. Brown

Keyword(s):

X Chromosome ◽

Peer Review Process ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

X Inactivation ◽

Chromosome Inactivation ◽

Xist Expression ◽

Inactivation Center ◽

Xist Rna ◽

Epigenetic Phenomenon

X-chromosome inactivation is a fascinating epigenetic phenomenon that is initiated by expression of a noncoding (nc)RNA, XIST, and results in transcriptional silencing of 1 female X. The process requires a series of events that begins even before XIST expression, and culminates in an active and a silent X within the same nucleus. We will focus on the role that transgenic systems have served in the current understanding of the process of X-chromosome inactivation, both in the initial delineation of an active and inactive X, and in the function of the XIST RNA. X inactivation is strictly cis-limited; recent studies have revealed elements within the X-inactivation center, the region required for inactivation, that are critical for the initial regulation of Xist expression and chromosome pairing. It has been revealed that the X-inactivation center contains a remarkable compendium of cis-regulatory elements, ncRNAs, and trans-acting pairing regions. We review the functional componentry of the X-inactivation center and discuss experiments that helped to dissect the XIST/Xist RNA and its involvement in the establishment of facultative heterochromatin.

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Single Cell Expression Data Reveal Human Genes that Escape X-Chromosome Inactivation

10.1101/079830 ◽

2016 ◽

Cited By ~ 2

Author(s):

Kerem Wainer-Katsir ◽

Michal Linial

Keyword(s):

X Chromosome ◽

Direct Method ◽

Single Cells ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

P Value ◽

Expression Data ◽

Rna Seq ◽

Specific Expression ◽

Biallelic Expression

ABSTRACTSex chromosomes pose an inherent genetic imbalance between genders. In mammals, one of the female’s X-chromosomes undergoes inactivation (Xi). Indirect measurements estimate that about 20% of Xi genes completely or partially escape inactivation. The identity of these escapee genes and their propensity to escape inactivation remain unsolved. A direct method for identifying escapees was applied by quantifying differential allelic expression from single cells. RNA-Seq fragments were assigned to informative SNPs which were labeled by the appropriate parental haplotype. This method was applied for measuring allelic specific expression from Chromosome-X (ChrX) and an autosomal chromosome as a control. We applied the protocol for measuring biallelic expression from ChrX to 104 primary fibroblasts. Out of 215 genes that were considered, only 13 genes (6%) were associated with biallelic expression. The sensitivity of escapees' identification was increased by combining SNP mapping for parental diploid genomes together with RNA-Seq from clonal single cells (25 lymphoblasts). Using complementary protocols, referred to as strict and relaxed, we confidently identified 25 and 31escapee genes, respectively. When pooled versions of 30 and 100 cells were used, <50% of these genes were revealed. We assessed the generality of our protocols in view of an escapee catalog compiled from indirect methods. The overlap between the escapee catalog and the genes’ list from this study is statistically significant (P-value of E-07). We conclude that single cells’ expression data are instrumental for studying X-inactivation with an improved sensitivity. Finally, our results support the emerging notion of the non-deterministic nature of genes that escape X-chromosome inactivation.

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Mitochondrial RNA granules are fluid condensates, positioned by membrane dynamics

10.1101/747055 ◽

2019 ◽

Author(s):

Timo Rey ◽

Sofia Zaganelli ◽

Emilie Cuillery ◽

Jean-Claude Martinou ◽

Suliana Manley

Keyword(s):

Dynamic Properties ◽

Mitochondrial Fission ◽

Super Resolution ◽

Membrane Dynamics ◽

Cellular Respiration ◽

Mitochondrial Rna ◽

Structured Illumination ◽

Inner Mitochondrial Membrane ◽

Structured Illumination Microscopy ◽

Rna Granules

Mitochondria contain the genetic information and expression machinery to produce proteins essential for cellular respiration. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins, and mitoribosome assembly factors are known to form punctate subcompartments referred to as mitochondrial RNA granules (MRGs) 1–3. Despite their proposed role in regulating gene expression, little is known about the structural and dynamic properties of MRGs. We investigated the organization of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy techniques, obtaining ultrastructural details of their internal architecture. We find that MRGs are organized into nanoscale RNA cores surrounded by a protein shell. Using live-cell super-resolution structured illumination microscopy and photobleaching perturbations, we reveal that MRGs undergo fusion and rapidly exchange components, consistent with liquid-liquid phase separation (LLPS). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with membrane remodeling. Inhibition of mitochondrial fission leads to an aberrant distribution of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our results reveal a role for LLPS in concentrating RNA and its processing proteins into MRGs, which are positioned along mitochondria by membrane dynamics.

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