Experimental observations of a nuclear matrix

Abstract

Nuclei are intricately structured, and nuclear metabolism has an elaborate spatial organization. The architecture of the nucleus includes two overlapping and nucleic-acid-containing structures - chromatin and a nuclear matrix. The nuclear matrix is observed by microscopy in live, fixed and extracted cells. Its ultrastructure and composition show it to be, in large part, the ribonucleoprotein (RNP) network first seen in unfractionated cells more than 30 years ago. At that time, the discovery of this RNP structure explained surprising observations that RNA, packaged in proteins, is attached to an intranuclear, non-chromatin structure. Periodic and specific attachments of chromatin fibers to the nuclear matrix create the chromatin loop domains that can be directly observed by microscopy or inferred from biochemical experiments. The ultrastructure of the nuclear matrix is well characterized and consists of a nuclear lamina and an internal nuclear network of subassemblies linked together by highly structured fibers. These complex fibers are built on an underlying scaffolding of branched 10-nm filaments that connect to the nuclear lamina. The structural proteins of the nuclear lamina have been well characterized, but the structural biochemistry of the internal nuclear matrix has received less attention. Many internal matrix proteins have been identified, but far less is known about how these proteins assemble to make the fibers, filaments and other assemblies of the internal nuclear matrix. Correcting this imbalance will require the combined application of biochemistry and electron microscopy. The central problem in trying to define nuclear matrix structure is to identify the proteins that assemble into the 10-nm filaments upon which the interior architecture of the nucleus is constructed. Only by achieving a biochemical characterization of the nuclear matrix will we advance beyond simple microscopic observations of structure to a better understanding of nuclear matrix function, regulation and post-mitotic assembly.

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

“…It is well-accepted that nuclear architecture provides a framework that is ultimately linked to the functional regulation of most, if not all, metabolic processes taking place in the mammalian cell nucleus. , In the 1970s, subnuclear organization was proposed to be dependent on the presence of a rigid structural framework. This putative nucleoskeleton, derived from nuclei that were subjected to DNase I digestion followed by ammonium sulfate or NaCl salt extraction, was commonly termed the nuclear matrix (NM) . Notably, the NM could be visualized only by negative staining electron microscopy (EM) after the removal of chromatin, which otherwise confined the ultrastructural characterization of the NM .…”

“…This putative nucleoskeleton, derived from nuclei that were subjected to DNase I digestion followed by ammonium sulfate or NaCl salt extraction, was commonly termed the nuclear matrix (NM) . Notably, the NM could be visualized only by negative staining electron microscopy (EM) after the removal of chromatin, which otherwise confined the ultrastructural characterization of the NM . Thus, several procedures that deplete nuclear chromatin while trying to preserve the NM architecture have been developed, revealing a filamentous meshwork spanning the inner nuclear space. − Apart from the nuclear intermediate filament proteins, named lamins (discovered in 1978), the constituents repeatedly identified in the insoluble nuclear material include a bewildering spectrum of proteins assigned to various functions, predominantly correlating with lamina formation, DNA repair, higher-order chromosome organization, genome replication, gene transcription, and RNA processing .…”

Identification of Evolutionarily Conserved Nuclear Matrix Proteins and Their Prokaryotic Origins

“…In contrast, the dynamic and complex internal architecture of the nucleus, first proposed to be supported by the nuclear matrix (NuMat), has not been incorporated in studies on nuclear evolution. Although various studies over the years have shown that the NuMat provides a structural framework for the functional regulation of most, if not all, nuclear metabolic processes such as DNA replication, transcription, splicing, DNA repair, and higher-order chromatin organization, − the in vivo existence of this structure has been widely debated due to concerns about the artefactual attachment of nuclear components during preparation. , Nonetheless, fluorescence microscopic techniques and RNA-selective staining procedures followed by electron microscopy have demonstrated a similar fibro-granular structure even in live or unextracted cells, supporting the idea of an in vivo NuMat. − The complication arises due to the fact that the biochemical composition of NuMat varies widely depending on extraction methods employed, despite their ultrastructural similarity, and has left the question of identifying the core components of NuMat unanswered. , This lack of annotation of the core nucleoskeletal proteins has led to the exclusion of NuMat composition from consideration in studies of nuclear evolution, impeding the progress of the field . Second, in recent years, attempts have been made to circumvent this drawback by genome-wide comparisons of extant eukaryotes.…”

“…13−17 The complication arises due to the fact that the biochemical composition of NuMat varies widely depending on extraction methods employed, despite their ultrastructural similarity, and has left the question of identifying the core components of NuMat unanswered. 17,18 This lack of annotation of the core nucleoskeletal proteins has led to the exclusion of NuMat composition from consideration in studies of nuclear evolution, impeding the progress of the field. 4 Second, in recent years, attempts have been made to circumvent this drawback by genome-wide comparisons of extant eukaryotes.…”

SAF-A mutants disrupt chromatin structure through dominant negative effects on RNAs associated with chromatin

“…Early studies identified a number of proteins that remain with insoluble nuclear material after removal of most chromatin and nuclear proteins, giving rise to the debated concept of a non-chromatin nuclear “scaffold” or “matrix”, which was thought to underpin nuclear chromosome structure (reviewed in (Nickerson 2001 )). One such protein thus identified was scaffold attachment factor A (SAF-A), so named because of its high affinity for certain sites on chromatin that resist nuclear extraction, suggested to be “scaffold attachment sites” (Fackelmayer et al 1994 ).…”