Formation of low molecular weight RNA species in HeLa cells

It has been previously shown that newly synthesized nuclear low molecular weight RNA species C and D are first detected in the cytoplasm for a few minutes before they are finally found in the nucleus. The following are some of the observations made in the present study, regarding the formation of C and D RNA: (1) The 5′ end cap ribose methylation of the C RNA precursor is complete in its cytoplasmic stage; the internal ribose methylation of the precursor seems to be completed about the time of its apparent transition from cytoplasm to nucleus. (2) The few nucleotides lost from the D RNA precursor during maturation seem to be excised sometime near its apparent cytoplasmic → nuclear transition. Newly synthesized C RNA also appears to lose some of its non-conserved nucleotides about the time of that transition, while the other extra nucleotides are lost later, in the nucleus. (3) The maturation of C and D RNA is inhibited early during suppression of protein synthesis by cycloheximide, while their synthesis is not. (4) The cytoplasmic precursors of C and D RNA are not associated with ribonucleoprotein particles as large as those reported for mature C and D RNA, although they do not appear to be free in the cytoplasm. (5) When the cellular UTP pool is depleted by exposure of the cells to amino sugars, and the synthesis of C, D, and other RNA species decreases, the level of[³H]uridine labeling of C and D RNA increases, while that of 4 S and 5 S RNA does not. These data are compatible with the existence of more than one nuclear UTP pool.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Presence of histone mRNA sequences in high molecular weight RNA of HeLa cells

Pulse-labeled HeLa cell RNA centrifuged under denaturing conditions was hybridized with DNA of recombinant phages containing sea urchin histone genes. This cross-hybridization showed the presence of histone mRNA sequences in high molecular weight RNA molecules. Treatment of the cells with actinomycin to stop RNA synthesis resulted in the rapid decay of this high molecular weight RNA followed by an increase of 9S histone mRNA in the cytoplasm.The results are consistent with the presence in HeLa cells of a high molecular weight precursor to histone messengers.     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

Synthesis of low molecular weight RNA components in cells with a temperature-sensitive polymerase II

AF 8 cells are a mutant cell line of baby hamster kidney cells with a temperature-sensitive polymerase II activity. When these cells grow at the non-permissive temperature (40 degrees C) the syntheseis of low molecular weight RNA components D, C and A is preferentially inhibited, whereas the synthesis of rRNA, tRNA, 5 S RNA and component L is affected only a little or not at all. These results indicate that polymerase II catalyzes the synthesis of components D, C and A.     

Bacteriophage phi 80-induced low molecular weight RNA

A species of low molecular weight RNA (M₃) which is produced in bacteriophage φ80-infected Escherichia coli has been isolated and characterized. M₃ appears immediately after infection and its synthesis continues until lysis. This RNA is unstable; its mean half-life is 13.5 min. The structural gene for M₃ is localized on the φ80 genome to the right of the exonuclease locus and to the left of gene Q.     

Low molecular weight RNA species from chromatin.

Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.     

Accumulation of low molecular weight DNA and changes in chromatin structure in HeLa cells treated with human fibroblast interferon

The addition of human fibroblast interferon (IFN-beta) (100 units/ml) at the S/G2 boundary of the cell cycle of synchronously grown HeLa cells is characterized by the accumulation of newly synthesized low molecular weight DNA and changes in chromatin assembly. In addition, there is a 3-fold stimulation in the incorporation of tracer amounts of [3H]thymidine, but not [3H] deoxyguanosine, into DNA and a 2-fold increase in the incorporation of [3H]dTTP into the DNA of isolated nuclei. Fluorescence-activated cell sorting by laser flow cytometry revealed that IFN-beta-treated cells were delayed in entering and passing through the S phase. The inhibition of proliferation of HeLa cells treated with IFN-beta is characterized by a 3-fold accumulation of newly synthesized DNA of Mr less than 56 X 10(6) compared to untreated cells as determined by alkaline sucrose gradient centrifugation. The newly synthesized DNA in IFN-beta-treated cells was replicative and not repair DNA. The observation that IFN-beta inhibits the processing of newly synthesized low molecular weight DNA into normal DNA might be explained by the intracellular accumulation of S-adenosylhomocysteine in IFN-beta-treated HeLa cells (de Ferra, F., and Baglioni, C. (1983) J. Biol. Chem. 258, 2118-2121) which could change the soluble ribonucleotide and deoxyribonucleotide pool and ultimately affect DNA processing. Interferon may also affect processing of DNA by interfering with normal chromatin assembly. Evidence for the effect of IFN-beta on chromatin assembly is provided; we have observed a more condensed structure in IFN-beta treated cells by circular dichroism spectroscopy. Simultaneous with the affect on chromatin assembly, there is a 70% decrease in poly(ADP-ribosylation) of either histone and/or non-histone proteins. The loss of coordination between the pool size for DNA synthesis, decreased postsynthetic modifications of chromatin, and normal chromatin formation may explain the inability of the cell to differentiate and to continue cell division.     

Intracellular distribution, assembly and effect of disease-associated connexin 31 mutants in HeLa cells

Mutations in connexin 31 （Cx31） are associated with erythrokeratodermia variabilis （EKV）,hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant （L34P） and four hearing impairment-associated mutants （66delD,141delI, R180X and E183K）, significantly reduced plaque formation in the cells with five EKV-associated dominant mutants （G12R, G12D, R42P, C86S and F137L） and no obvious change in the cells with two other mutants （I 141V and 652del 12）. Immunoblotting analysis showed that 12 mutated Cx31 s, like WTCx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants （G12R, G12D, R42P, C86S and F137L） caused cell death in HeLa cells. However, the five heating impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, diseaseassociated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.     

Rat liver nuclear skeleton and small molecular weight RNA species

Small molecular weight RNA species (smwRNAs) were studied in rat liver nuclei with and without chromatin as well as with and without nuclear envelope and nucleoplasm. From all the species identified, only two, N5 and 5Sb, were related to ribosomes. The others were localized exclusively in the nuclear skeleton or the spongelike network that was described in the preceding communication. This network or protein matrix contains a less abundant but exclusive set of molecules designated 5Sa, N1, and 4.5S, as well as other more abundant molecules which also exist in rat liver endoplasmic reticulum but not in polysomes or postribosomal RNP complexes. The smwRNAs behave like HnRNA; they remain located in the nuclear skeleton when nuclei are deprived of nucleoplasm and chromatin. With the information presently available, it is not possible to know whetherer both species are in the same or different RNP complexes and whether some of the smwRNAs contribute to the architecture of the nuclear skeleton. Distinct from any other nuclear RNA species, smwRNAs have two unique properties: facility of extraction, and resistance to nuclear ribonuclease digestion.