Cellular Skeletons and RNA Messages
Publisher Summary
This chapter presents the experimental results that suggest an interrelation between cellular topology and nucleic acid metabolism. Gentle lysis of HeLa cells with a nonionic detergent reveals an elaborate remnant structure that retains much of the cellular morphology. Electron micrographs and biochemical studies show that a majority of the protein synthetic apparatus remains affixed to this “cytoskeleton.” It discusses that the skeleton structure possibly plays an important role in cytoplasmic RNA metabolism. Two of the small RNA species found in mammalian cells, ScK and ScL, are intimately associated with the cytoskeleton, and ScL is, in part, associated with membranes. The function of these molecules is unknown, but there is the possibility that they serve a structural role and may help determine the architecture of the cytoskeleton. If so, then RNA metabolism could play a direct role in determining cellular topology. The nucleus also has a “skeletal” structure, although much less can be visualized in this case. Many of the small RNA species are found localized in this nuclear skeleton, suggesting that they also may play a role in either structure or function determination. The chapter reviews that the experiments indicates an aspect of the metazoan cell with no obvious counterpart in bacteria—that is, an interrelation between spatial organization and RNA metabolism.
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Cited by (21)
The Nuclear Matrix: A Structural Milieu for Genomic Function
1996, International Review of CytologyWhile significant progress has been made in elucidating molecular properties of specific genes and their regulation, our understanding of how the whole genome is coordinated has lagged behind. To understand how the genome functions as a coordinated whole, we must understand how the nucleus is put together and functions as a whole. An important step in that direction occurred with the isolation and characterization of the nuclear matrix. Aside from the plethora of functional properties associated with these isolated nuclear structures, they have enabled the first direct examination and molecular cloning of specific nuclear matrix proteins. The isolated nuclear matrix can be used for providing an in vitro model for understanding nuclear matrix organization in whole cells. Recent development of high-resolution and three-dimensional approaches for visualizing domains of genomic organization and function in situ has provided corroborative evidence for the nuclear matrix as the site of organization for replication, transcription, and post-transcriptional processing. As more is learned about these in situ functional sites, appropriate experiments could be designed to test molecular mechanisms with the in vitro nuclear matrix systems. This is illustrated in this chapter by the studies of nuclear matrix-associated DNA replication which have evolved from biochemical studies of in vitro nuclear matrix systems toward three-dimensional computer image analysis of replication sites for individual genes.
Differential behavior of liposome-introduced specific RNAs in living Drosophila cells
1988, Gene Analysis TechniquesWe have developed a protocol for efficiently introducing macromolecules into Drosophila tissue culture cells using liposomes. By carefully adjusting the fusion parameters, conditions have been established to routinely encapsulate 15–30% of the starting material into liposomes and to introduce 20–30% of the liposome-encapsulated material into the cells during a 30-minute fusion period. Essentially, all of the cells receive material from the liposomes and 109 cells can be fused at once. The fusion does not have any measureable effect on cell viability as assayed by trypan blue exclusion, growth rate, and cell morphology. We have utilized this technique to introduce radioactive RNAs into nonradioactive cells, thus enabling the behavior of the introduced RNAs to be followed unambiguously. Liposome-introduced small nuclear RNAs (snRNAs) are stable in the cell for at least 25 hours (approximately two cell generations), with 80% of the radioactivity remaining trichloroacetic acid (TCA) precipitable and the gel electrophoresis pattern remaining essentially unchanged. This is in contrast to liposome-introduced cytoplasmic RNAs, which are only 20% TCA precipitable after the first hour. In the cell, the introduced snRNAs attain a 10–35-fold higher concentration in the nucleus than the cytoplasm. Nuclear accumulation is not seen with Drosophila tRNA or 5s RNA, both of which attain the same nuclear as cytoplasmic RNA concentration.
Thermal diminution and augmentation of the retention of transportable rRNA in nuclear envelope-free nuclei
1984, BBA - Gene Structure and ExpressionWe have examined the effect of temperature on the rRNA transport from nuclei isolated from Tetrahymena after removal of both nuclear membranes and pore complexes by 1% Triton X-100. These nuclei export rRNA as precursor ribosomal ribonucleoprotein particles at both 28°C and 8°C which are qualitatively the same in terms of rRNA pattern, sedimentation coefficients and buoyant densities. At 8°C, however, significantly fewer ribosomal ribonucleoprotein particles can be maximally exported than at 28°C, though nuclei contain enough potentially transportable particles. These are increasingly released with increasing temperatures. Under conditions non-permissive for export, temperature elevation decreases the number of the potentially transportable ribosomal ribonucleoprotein particles in nuclei. Our data show: (i) transportable ribosomal ribonucleoprotein particles inside nuclei are not ‘free’, but rather are subject to a complex temperature-sensitive retention: this retention is gradually diminished under export conditions and augmented under non-permissive export conditions with increasing temperatures. (ii) These retention mechanisms operate at an intranuclear level preceding the ribosomal ribonucleoprotein passage through the nuclear envelope pore complexes, i.e., the nuclear envelope regulates neither the number of potentially transportable ribosomal ribonucleoprotein particles in nuclei nor the number of those particles which can be maximally exported from nuclei at a given temperature. We suggest that these retention mechanisms involve temperature-sensitive domains of the nuclear matrix.
Heterogeneity and Territorial Organization of the Nuclear Matrix and Related Structures
1983, International Review of CytologyThis chapter discusses the heterogeneity and territorial organization of the nuclear matrix and related structures. Nonchromatin structures (NCSs) are structures containing RNA, such as the perichromatin and interchromatin fibrils, granules and nuclear bodies, together with certain nucleolar components, nuclear membrane material, and pore complexes. Several roles of NCS in connection with the maintenance of nuclear organization include (1) the coiling of DNA molecules into superhelical loops, (2) the segregation of chromatin into condensed and diffuse zones, and (3) the positioning of distinct chromosome segments in the nuclear volume. Genetically active sequences occupy definite positions along the DNA loops. The direct involvement of NCSs in gene regulation would only be possible when active genes are located close to the attachment points of the loops. 3D electron microscopy constitutes a powerful approach to verify the existence and distribution of NCSs.
Mild nuclease treatment as a probe for a non-random distribution of adenovirus-specific RNA sequences and of cellular RNA in nuclear ribonucleoprotein fibrils
1982, Journal of Molecular BiologyAt the late period of productive infection, the RNA of hnRNP† from adenovirus-infected HeLa cells contains primarily cellular RNA and the partially processed transcripts from the major late transcription unit. Total cellular and viral RNA, seven viral RNA fragments corresponding to messenger and non-messenger RNA sequences, were determined in hnRNP. A mild nuclease treatment of 30 to 200 S native hnRNP, sufficient for the separation of 25 to 55 S monoparticles from larger (100 to 200 S) RNP, served as a probe for hnRNP structure and allowed the study of sequence distribution in these two classes of hnRNP units. The main observations were as follows: (1) 90% of the total viral RNA initially present in large hnRNP was found in the large RNP after nuclease treatment, whereas 85% of the nuclear-restricted cellular RNA was released with monoparticles; (2) viral poly(A)+ RNA was enriched in large RNP indicating an enrichment in viral premessenger RNA; (3) the viral-specific sequences were distributed non-randomly between the hnRNP units. This distribution did not depend on their nucleotide sequence (messenger or non-messenger nature). Rather, the sequences from the 5′ half of the transcription unit were enriched in large RNP and those from the 3′ half in monoparticles. This may be related to the presence, in addition to authentic premessenger RNA, of nuclear-restricted hnRNA synthesized from the 3′ part of the transcription unit after the polyadenylation sites (Nevins & Darnell, 1978). The preferential localization of premessenger RNA in the large RNP isolated after mild nuclease treatment suggested that these structures are the sites of splicing. The monoparticles that are enriched in nuclear-restricted sequences might be structures where degradation starts.
The nuclear matrix of duck erythroblasts is associated with globin mRNA coding sequences but not with the major proteins of 40S nuclear RNP
1981, Experimental Cell ResearchA residual protein matrix has been prepared from avian erythroblast nuclei by extensive extraction with salines and detergent and subsequent digestion with high concentrations of RNase and DNase. Ultrastructural examination reveals considerable internal structure, the most prominent feature being the remains of the nucleoli embedded in a network of fibres of fairly uniform diameter of 50 Å. The proteins which make up this structure have been examined by two-dimensional electrophoresis and are shown to consist of a characteristic set of about 30, mainly acidic components, including four prominent species of 43 000, 52 000, 66 000 and 68 000 molecular weight (MW). In parallel preparations of the nuclear matrix digested with DNase alone, much of the nuclear RNA is found associated with the residual structure, including globin-coding sequences. These results correlate well with the ultrastructural appearance of DNase-digested matrix preparations which show that superimposed on the 50 Å fibrous network is a 200–300 Å granular component, the combined fibrillo-granular structure resembling the interchromatin RNP previously identified in situ. However, the proteins of the DNase-digested matrix seen by two-dimensional electrophoresis are indistinguishable from the proteins of matrix preparations digested with both DNase and RNase. Furthermore, two-dimensional comparison between the proteins of the DNase-digested matrix and purified 40S nuclear RNP particles shows that the bulk of the proteins found associated with nuclear RNA in vitro are extracted during matrix preparation, and only two, with MWs of 43 000 and 73 000, remain. The latter species co-migrates with the poly(A)-binding protein.