Sheldon Penman

Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and polyribosome-associated messenger RNA in HeLa cells☆

Abstract Three heterogeneous RNA species in HeLa cells have been characterized with respect to kinetics of labeling, turnover and location. These are the nucleoplasmic, cytoplasmic and polyribosome-associated messenger fractions. The heterogeneous nucleoplasmic RNA is found entirely in the nucleoplasm if sufficently high ionic strength buffer and zonal sucrose density centrifugation are used in the nuclear fractionation. The RNA is isolated in the form of particulates with sedimentation coefficients up to 5000 s. Nucleoplasmic RNA synthesis is shown to be relatively unaffected by doses of actinomycin which completely inhibit ribosomal RNA synthesis. The labeling of the nucleoplasmic RNA indicates that it turns over rapidly with a mean life of approximately one hour. The rate of decay of the nucleoplasmic RNA in the presence of a high concentration of actinomycin is initially similar to the rate of formation of this RNA, but subsequently becomes aberrant. A heterogeneous RNA is found associated with the cytoplasm using the fractionation techniques described. Its kinetics of labeling suggest that it is unrelated to the nucleoplasmic RNA. Only a portion of the total cytoplasmic RNA is associated with polyribosomes. Polyribosomes are unaffected by the level of actinomycin used to inhibit ribosomal RNA synthesis for at least seven hours. Labeled messenger RNA can be isolated from polyribosomes free of contamination by newly synthesized ribosomal RNA. A heterogeneous RNA which co-sediments with, but is not attached to, polyribosomes is identified. Polyribosome-attached messenger RNA has a range of sedimentation values from 8 to 30 s with a maximum at about 18 s, whereas the contaminating RNA is broadly distributed from 10 to 70 s. Agents which alter the sedimentation of polyribosomes in preparative sucrose gradients, such as puromycin and EDTA, also alter the distribution of messenger RNA in a similar manner but leave the non-messenger RNA unaffected. The kinetics of appearance of messenger RNA indicate that it is probably not derived from the nucleoplasmic heterogeneous RNA. The existence of a pool is inferred which results in a 15-minute lag before labeled messenger RNA appears on polyribosomes, and which continues to supply messenger for 20 minutes after the administration of a high concentration of actinomycin.

A cytoskeletal structure with associated polyribosomes obtained from HeLa cells

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cells ultrastructural features, such as 100A filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100A filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.

Small molecular weight monodisperse nuclear RNA

Abstract The nuceloplasm and nucleolus of HeLa cells contain six distinct low molecular weight species of RNA. Their size, as estimated from electrophoretic mobility, ranges from 100 to 180 nucleotides. These RNA species are long-lived and do not appear to be precursor to any cytoplasmic product. At least four of them are extensively methylated. Their base compositions range from 47 to 54% guanosine + cytidine. Similar species of nuclear RNA are found in mouse fibroblast cultures and in the developing chick embryo brain.

The nuclear matrix: three-dimensional architecture and protein composition.

The structural filament network of the nucleus is prepared while still connected to the cytoskeleton. The relatively gentle procedure removes about 98% of the DNA and at least 86% of the histones. The matrix is bounded by an outer nuclear lamina connected to the cytoskeletal framework, as well as the inner filaments. The filaments range in diameter from 3 to 22 nm, and are organized in a three-dimensional anastomosing network in which nucleoli are enmeshed. The nuclear matrix is separated from the cytoskeletal framework by a double detergent and then partitioned into a chromatin fraction and a matrix fraction by nuclease and high salt. Two-dimensional gel electrophoresis shows that the proteins of the cytoskeleton, chromatin and nuclear matrix are very different. A major protein found in all fractions cofocuses with actin. Vimentin is largely associated with the nuclear matrix, probably as a corona external of filaments.

Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts

Anchorage-dependent mouse fibroblasts grow only if attached to and spread on a solid substrate. The suspension of cells in methyl-cellulose results in dramatic, coordinated inhibition of the major RNA and protein synthesis systems, and these systems are sequentially restored when cells are replated on a tissue culture dish surface. In the present report the effects on metabolism of cell reattachment are separated from those of subsequent spreading by controlling cell shape. Macromolecular metabolism is first strongly suppressed by long-term suspension culture. The cells are then replated in the presence of a variety of spreading inhibitors. The recovery of protein synthesis, which rapidly follows reattachment, does not require extensive cell spreading. Contact of a limited portion of the plasma membrane with the solid culture dish surface is apparently a sufficient signal by itself. A very different method of controlling cell shape is afforded by changing culture dish surface adhesivity. Suspended cells are replated on dishes precoated with thin layers of the hydrophilic hydrogen poly(2-hydroxyethyl methacrylate). The final mean cell diameter is then varied over wide limits. As before, protein synthesis recovery is unaffected. However, nuclear events such as DNA and rRNA synthesis and mRNA production are profoundly affected by cell shape. Thus, cell surface contact and cell shape give rise to distinctly different regulatory responses.

Regulation of protein synthesis in mammalian cells: II. Inhibition of protein synthesis at the level of initiation during mitosis☆☆☆

Abstract The decline in the rate of protein synthesis during mitosis in eukaryotic cells was studied using Chinese hamster ovary cells arrested in metaphase with Colcemid. The rate of protein synthesis in mitotic cells is approximately 30% of the interphase rate. Ribosomes become attached to messenger RNA and initiate polypeptide synthesis at a reduced rate. After initiation of polypeptide synthesis, ribosomes are translated at a normal rate. Reducing the rate of translation with low levels of cycloheximide mobilizes a large proportion of monomeric ribosomes into active polyribosomes. The amount of messenger RNA associated with polyribosomes is also increased in mitotic cells treated with cycloheximide. It appears that the reduced protein synthesis during mitosis results from a lowered rate of attachment of ribosomes to messenger RNA and initiation of polypeptide synthesis.

Nestin expression in hair follicle sheath progenitor cells

The intermediate filament protein, nestin, marks progenitor cells of the CNS. Such CNS stem cells are selectively labeled by placing GFP under the control of the nestin regulatory sequences. During early anagen or growth phase of the hair follicle, nestin-expressing cells, marked by GFP fluorescence in nestin-GFP transgenic mice, appear in the permanent upper hair follicle immediately below the sebaceous glands in the follicle bulge. This is where stem cells for the hair follicle outer-root sheath are thought to be located. The relatively small, oval-shaped, nestin-expressing cells in the bulge area surround the hair shaft and are interconnected by short dendrites. The precise locations of the nestin-expressing cells in the hair follicle vary with the hair cycle. During telogen or resting phase and in early anagen, the GFP-positive cells are mainly in the bulge area. However, in mid- and late anagen, the GFP-expressing cells are located in the upper outer-root sheath as well as in the bulge area but not in the hair matrix bulb. These observations show that the nestin-expressing cells form the outer-root sheath. Results of the immunohistochemical staining showed that nestin, GFP, keratin 5/8, and keratin 15 colocalize in the hair follicle bulge cells, outer-root sheath cells, and basal cells of the sebaceous glands. These data indicate that nestin-expressing cells, marked by GFP, in the hair follicle bulge are indeed progenitors of the follicle outer-root sheath. The expression of the unique protein, nestin, in both neural stem cells and hair follicle stem cells suggests their possible relation.

Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells

When the cytoskeletal framework is prepared from suspension-grown HeLa by extraction with nonionic detergent, all the polyribosomes are associated with the framework while 80% of tRNA and the major portion of monoribosomes as well as 75% of the cell proteins are found in the soluble fraction. The mRNA of polyribosomes is bound to the cytoskeleton and these molecules remain attached even after polyribosomes are disassembled in vivo prior to extraction. Although all actively translating message molecules are attached to the framework, about one quarter of the poly(A)+ mRNA is free of the framework. The binding of message to the skeleton may be obligatory for translation. Upon infection with VSV, all the viral polyribosomes but not all the viral messages of the infected cell are associated with the cytoskeletal framework. Pulse-chase labeling shows that VSV messages initially associate with the framework and then later detach and cease translation. The mRNA for the viral glycoprotein (G), known to translate only on ribosomes bound to endoplasmic reticulum, is also retained by the detergent-extracted structure. It appears that the protein substructure of the endoplasmic reticulum which binds polyribosomes is a component of the cytoskeletal framework.

Messenger RNA in HeLa Cells : Kinetics of Formation and Decay

Abstract The polyadenylic acid-containing messenger RNA fraction of HeLa cells was measured by its affinity for oligedeoxythymidylate cellulose. Both the kinetics of initial labeling and the decay after a brief pulse of incorporation were examined. The kinetics of decay are complex, but can be approximated by assuming two populations; a short-lived species with a half-life of seven hours and a long-lived component with a half-life of 24 hours. It is estimated that the short-lived material comprises 33% of total cellular mRNA, while the relatively stable species amounts to 67% of the steady-state mRNA content. The two mRNA components with different decay times were observed simultaneously in the same cell population by measuring decay of 24-hour old mRNA labeled with 14C and RNA briefly labeled with 3H. The old mRNA had only a 24-hour decay component, while the new mRNA was biphasic. The decay of old and new mRNA was also observed after RNA synthesis was inhibited with actinomycin. Again, old mRNA decayed more slowly than recently labeled material. However, both decay times are significantly shorter in the presence of actinomycin and correspond to half-lives of approximately 4 and 12 hours. There is a small but significant difference in sedimentation distribution of new and old mRNA, the old mRNA sedimenting more slowly than new material, suggesting that the more stable species has a lower average molecular weight. The steady-state content of mRNA in HeLa cells amounts to 5.5% of the ribosomal RNA, or more than twice the amount of messenger RNA estimated to be on hemoglobin-synthesizing polyribosomes.

Methylation and processing of ribosomal RNA in HeLa cells

The methylation of RNA in HeLa cells has been studied. After incorporation of radioactive methyl-labeled methionine, the cells were fractionated into cytoplasmic, nuclear, and nucleolar fractions. Methylation of high molecular weight RNA is shown to be exclusively in the 45 s ribosomal RNA precursor and takes place in the nucleolus. The methylation of the 45 s RNA apparently occurs close to the growing point of newly synthesized RNA as evidenced by the existence of methylated, partially completed molecules after very short pulse labeling. The methyl-labeled RNA was chased with non-radioactive methionine and the progression of the ribosomal RNA precursors was followed through the cell fractions. The data support the previously published hypothesis that nucleolar 45 s RNA is the precursor to cytoplasmic 18 s RNA and 32 s nucleolar RNA. The 32 s RNA is converted to 28 s RNA, which is then found in the nucleoplasm and is the apparent precursor to cytoplasmic 28 s RNA.