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Subfractionation of Nuclear Matrix (NuMat)

ABSTRACT: NuMat is a residual structure resistant to solubilization by nucleases, detergents, and salt solutions that retains the morphology of the nucleus. Purported to be the basis of nuclear architecture, it has remained further undissected. We use a chaotrope to subfractionate the nuclear matrix and decipher its organization.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Embryo

SUBMITTER: Ashish Bihani

LAB HEAD: Rakesh Kumar Mishra

PROVIDER: PXD031296 | Pride | 2023-10-25

REPOSITORIES: Pride

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	ESC-HF-1A1-100920-Ashish.raw	Raw
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Publications

Biochemical Deconstruction and Reconstruction of Nuclear Matrix Reveals the Layers of Nuclear Organization.

Bihani Ashish A Avvaru Akshay K AK Mishra Rakesh K RK

Molecular & cellular proteomics : MCP 20231019 12

Nuclear matrix (NuMat) is the fraction of the eukaryotic nucleus insoluble to detergents and high-salt extractions that manifests as a pan-nuclear fiber-granule network. NuMat consists of ribonucleoprotein complexes, members of crucial nuclear functional modules, and DNA fragments. Although NuMat captures the organization of nonchromatin nuclear space, very little is known about components organization within NuMat. To understand the organization of NuMat components, we subfractionated it with i ...[more]

PMID: 37863319

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Drosophila melanogaster nuclear matrix proteome

Project description:Nucleus is a highly structured organelle and contains many functional compartments. While the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we find structural proteins, chaperones related, DNA/RNA binding, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. Comparison of the 2D profile of NuMat proteome from different developmental stages of Drosophila embryos shows that less than half of the NuMat proteome is constant and rest of the proteins are stage specific dynamic components. This NuMat dynamics suggests a possible functional link between NuMat and the embryonic development. Finally, we also show that a subset of NuMat proteins remain associated with the mitotic chromosomes implicating their role in mitosis and possibly the epigenetic cellular memory. NuMat proteome analysis provides tools and opens up ways to understand nuclear organization and function.

2011-05-05 | PRD000255 | Pride

Fixed and unalterable Matrix Associated Regions in Drosophila melanogaster genome

Project description:Eukaryotic genome is compartmentalized into structural and functional domains. One of the concepts of higher order organization of chromatin posits that the DNA is organized in constrained loops that behave as independent functional domains. A predominantly ribo-proteinaceous nucleoskeleton, termed as Nuclear Matrix (NuMat) is proposed to provide the structural platform for attachment of these loops. The DNA sequence located at the base of the loops are known as the Matrix Attachment Regions (MARs). NuMat relates to all nuclear processes and has been shown to be cell type specific in composition. It is a biochemically defined structure and several protocols have been used to isolate the NuMat where some of the steps have been critically evaluated. In the present study we have looked into the dynamics of MARs when the isolation process is varied and also during embryonic development of D. melanogaster. Our results show that a subset of MARs termed here as “Core-MARs” are fixed and unalterable anchor points in the Drosophila genome as they remain associated with NuMat at all developmental stages and do not depend on the isolation procedure used. Core-MARs are abundant in the pericentromeric heterochromatin. On the other hand, MARs in the euchromatin are dynamic and reflect the transcriptomic profile of the developmental stage of the host cell. New MARs are generated by nuclear stabilization (a critical step in the isolation procedure), and during development, mostly at the paused RNA polymerase II (Pol II) promoters. Paused Pol II MARs depend on RNA transcription for NuMat association. RNase A treatment leads to collapse of the NuMat and loss of paused Pol II promoter MARs. Our data reveals the role of MARs in functional compartmentalization of D. melanogaster genome and adds to the current understanding of nuclear architecture and 3D organization of a functionally dynamic nucleus.

2022-10-25 | PXD031279 | Pride

Identification of evolutionarily conserved nuclear matrix proteins and their prokaryotic origins

Project description:Compared to prokaryotic cells, a typical eukaryotic cell is much more complex along with its endomembrane system and membrane-bound organelles. Although the endosymbiosis theories convincingly explain the evolution of membrane-bound organelles such as mitochondria and chloroplasts, very little is understood about the evolutionary origins of the nucleus, the defining feature of eukaryotes. Most studies on nuclear evolution have not been able to take into consideration the underlying structural framework of the nucleus known as the nuclear matrix (NuMat), a ribonucleoproteinaceous structure. This can largely be attributed to the lack of annotation of its core components. Since, NuMat has been shown to provide a structural platform for facilitating variety of nuclear functions such as replication, transcription, and splicing, it is important to identify its protein components to better understand these processes. In this study, we address this issue using the developing embryos of D. melanogaster and D. rerio and identify 362 core NuMat proteins that are conserved between the two organisms. We find that of them, 68 proteins are conserved across all eukaryotes and, therefore, have been indispensable for nuclear function for over 1.5 billion years of eukaryotic history. We also traced the prokaryotic origins of a significant proportion of the core NuMat proteins which paves the way to understand the evolution of nuclear architecture and functions.

2021-01-26 | PXD012926 | Pride

Nuclear scaffold and matrix attachment of HeLa chromatin

Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in HeLa cells. LIS or 2 M NaCl solutions followed by restriction digestion with EcoR1 facilitates the separation from scaffold/matrix bound DNA from non bound DNA. Genomic CGH arrays were used to map the relative differences between attached (scaffold/matrix) and non-attached (loop) portions of HeLa DNA. The expression profile of the HeLa cells used for aCGH analysis was also determined.

2010-05-22 | E-GEOD-13774 | biostudies-arrayexpress

Nuclear scaffold and matrix attachment of Aortic Adventitial Fibroblast (AoAF) chromatin

Project description:The nuclear scaffold/matrix provides an anchor for higher order genome structure that has both structural and functional implications. Different extraction protocols, i.e., utilizing either 25 mM LIS or 2 M NaCl, isolate somewhat different protein constituents of either the nuclear scaffold or nuclear matrix respectively. We have mapped, by array CGH, the locations of attachment to each of these residual protein bodies relative to non-attached DNA along the entire length of human chromosomes 14, 15, 16, 17 and 18 in AoAF cells. LIS (lithium 3,5-diiodosalicylate) or 2 M NaCl solutions followed by restriction digestion with EcoR1 facilitates the separation from scaffold/matrix bound DNA from non bound DNA. Genomic CGH arrays were used to map the relative differences between attached (scaffold/matrix) and non-attached (loop) portions of AoAF DNA. The expression profile of the AoAF cells used for aCGH analysis was determined.

2009-03-10 | E-GEOD-13792 | biostudies-arrayexpress

Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells – Transmission of hallmarks of nuclear organization through mitosis

Project description:During open mitosis chromatin condenses to form chromosomes and decondenses post-mitotically to re-occupy their designated nuclear territory in a precisely lineage specific manner. This necessitates that features of nuclear architecture persist through mitosis. Here we present a proteomic study, which shows that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosome. Proteomic comparison was made between nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an identical biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that a large graction of the NuMat proteins are retained in the MiCS and possibly play an important role in maintenance of cell lineage specific features of nuclear architecture during cell division and thereby serve as components of cellular memory.

2018-07-17 | PXD008404 | Pride

Impact of Surfactants on Cumulative Trypsin Activity for Bottom-up Proteome Analysis

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2024-04-23 | PXD050154 | Pride

Genome-wide chromatin profiles of PBRM1 [RNA-Seq]

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