High Molecular Weight Nuclear RNA Research Articles

Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo

The psoralen derivative 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) reacts with base-paired regions of RNA and forms interstrand covalent cross-links. Since psoralens permeate living cells, they can be used to probe RNA-RNA interactions in vivo. We used AMT to investigate whether small nuclear RNAs are base-paired with high molecular weight nuclear RNA in the cell. Intact HeLa cells were treated with AMT, and high molecular weight RNA was isolated under denaturing conditions from nuclei or from subnuclear fractions. The presence of base-paired snRNAs in the high molecular weight nuclear RNA was examined by electrophoresis after photochemical reversal of the cross-links. We found snRNA U3 and 5.8S rRNA to be cross-linked to nucleolar RNA. In contrast, snRNA U1 was cross-linked to high molecular weight RNA in ribonucleo-protein particles containing hnRNA. The U1 base-paired to hnRNA was identified by its hybridization with a cloned U1 DNA sequence after reversal of the cross-links. These results demonstrate that U1 is base-paired with hnRNA in vivo, suggesting a role in mRNA processing.

Complex molecular structure of the gene coding for rat ceruloplasmin

The distribution of ceruloplasmin-coding sequences among the fragments of rat nuclear DNA obtained after the complete digestion with seven restriction endonucleases ( EcoRI, BamHI, BspI, HindIII, KpnI, BglII and XhoI) was studied using highly specific cDNA probes. Although only a single copy of this gene per rat haploid genome was detected in DNA-cDNA hybridization in solution, the number of restriction fragments carrying the sequences of ceruloplasmin (CP) gene varied from two to five, depending upon the enzyme used, and their total length was several times higher than the minimal length of CP-coding gene, as deduced from the size of mRNA (2.3 Md for double-stranded DNA). The partial double-stranded DNA transcript of ceruloplasmin mRNA coding for about 70% of its length (from 3'-end) does not contain recognition sites for some restriction endonucleases generating multiple fragments of CP gene in cellular DNA. These data are consistent with a split pattern of CP gene which seems to consist of several exons and introns. The partial protection from Si nuclease of discrete fragments of full-length cDNA after annealing with high molecular weight nuclear RNA is consistent with this assumption and seems to be an indication that exons and introns are joined into a functional unit coding for high mol. wt. CP pre-mRNA.

The 5' terminal capping of heterogeneous nuclear RNA at different embryonic stages of the sea urchin.

5' Terminal cap structures of hnRNA have been characterized and the extent of capping determined as a function of embryonic development. Sea urchin embryo hnRNA contains only the type-1 cap, m7GpppNmpNp, with the type-2 cap, which has a 2'-0-methylated subpenultimate nucleotide, being associated only with stable small nuclear RNAs. These cap 2-containing RNAs are synthesized at a rate of approximately 70 molecules min-1 nucleus-1 compared to approximately 1000 molecules for hnRNA cap 1. Approximately 70% of nuclear cap 1 is associated with greater than 15S RNA in denaturing solvent, but under non-denaturing conditions the percentage is much higher. Cap 1 in low and high molecular weight nuclear RNA have the same kinetics of methyl labeling. Thus all cap 1 structures may belong to a single class either covalent or H-bonded to high molecular weight RNA. hnRNA greater than 15S is 35% capped; however, adding caps in less than 15S RNA gives an estimate of 50% capping for total hnRNA. In development from early blastula to late gastrula, there is little if any change in the extent of capping of hnRNA. These results coupled with others indicate that the fraction of hnRNA molecules serving as precursor to mRNA does not change quantitatively during embryonic development.

Polyoma virus high molecular weight nuclear rna codes for capsid protein VP2 in vitro

RNA extracted from the nuclei of 3T6 mouse cells late after infection by pot yoma virus (Py) has been translated in vitro to give one predominant polypeptide, which was identified as virion protein VP2 by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide analysis. The active nuclear RNA species sediment between 15 and 50 S on sucrose gradients after denaturation, and resediment true after further denaturation and recentrifugation on 50% formamide-sucrose gradients. The bulk of the active viral nuclear RNA does not bind to oligo(dT)-cellulose and synthesis of Py VP2 is inhibited by m 7Gp under conditions where translation of Py-cRNA and EMC RNA are not inhibited. These experiments suggest that large viral nuclear RNA molecules, which are probably capped but not polyadenylated, can serve as messengers for Py VP2.

The low molecular weight of RNAs of adenovirus 2-infected cells

The cytoplasm of HeLa cells infected with adenovirus type 2 contains many species of low molecular weight RNA, including several of viral origin. In addition to a 9 S messenger RNA, the viral genome gives rise to two species of virus-associated RNA: the major species is 5.5 S RNA or virus-associated RNA I, and the minor species is 5.2 S RNA or virus-associated RNA II. Virus-associated RNA I occurs in the cytoplasm in several electrophoretically separable forms, and its sequences are also present in high molecular weight nuclear RNA but not in cytoplasmic mRNA. The structure of virus-associated RNA II is shown to be distinct from that of the major species, and the position of its gene is mapped on the viral genome. The two virus-associated RNA genes are located on the r strand near position 30 of the adenovirus type 2 physical map, and are separated by a spacer of about 75 base-pairs.

Isolation of a distinct pool of polyribosomes from nuclei of adenovirus-infected HeLa cells

A separate and distinct population of polyribosomes exists in the detergent-washed nuclei of adenovirus-infected HeLa cells. These polyribosomes, released by exposure to polynucleotides such as high molecular weight nuclear RNA or poly(U), do not appear to be cytoplasmic contaminants. Nuclear polyribosomes have a considerably lower buoyant density compared to cytoplasmic ones. Nuclear polyribosomes, in a cell-free system of protein synthesis, are six- to eight-fold less active compared to cytoplasmic ones and are insensitive to aurin tricarboxylic acid. They do not complement cytoplasmic polyribosomes in protein synthesis in the cell-free system. Finally, the number of proteins synthesized by nuclear polyribosomes is higher compared with that synthesized by the cytoplasmic ones. Only the virus-specific proteins, including P-VII, are synthesized by cytoplasmic polyribosomes. Nuclear polyribosomes, on the other hand, synthesize virusspecific proteins, including P-VII and VII, and a number of additional proteins not synthesized by the cytoplasmic ones.

Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate.

Severe degradation of high molecular weight RNA was shown to occur during incubation with commercially purified DNase. Most of the RNase activity could be removed by passage of the DNase through a column of agarose-coupled amino phenylphosphoryl-uridine-2' (3')-phosphate. Incubation with the treated DNase caused only minimal alteration of the sedimentation pattern of high molecular weight nuclear RNA, determined under partially denturing conditions. No impairment of DNase activity was detected.

Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells

Highly purified steady state heterogeneous nuclear RNA from HeLa cells has been prepared by a new procedure. Detergent-washed nuclei are disrupted in 0.4 M ammonium sulfate, which also dissociates contaminating polysomes. The hnRNA remains bound to chromatin, which can be pelleted by gentle centrifugation. Ribonuclease inhibitors permit the preparation of very high molecular weight nuclear RNA. The hnRNA was cleaved with alkali. The poly(A)-containing fragments were separated from those containing oligo(A), and a cDNA copy was prepared. Hybridization of this nuclear cDNA to cytoplasmic mRNA showed that the scarce (complex) message sequences make up a larger proportion of nuclear RNA than of cytoplasmic RNA. In addition, at least 30% of the poly(A)-adjacent sequences in nuclear RNA have no apparent counterparts in the cytoplasm. cDNA prepared from hnRNA sedimenting faster than 45S under denaturing conditions gives similar results, showing the presence of both message and non-message sequences in very large transcripts. cDNA complementary to mRNA was separated into the abundant and scarce sequences, and hybridized separately to the poly(A)-adjacent sequences in nuclear RNA. The hybridization of the abundant sequence cDNA was used to set an upper limit on possible cytoplasmic contamination. Hybridization of the scarce sequence cDNA shows that nearly all the scarce cytoplasmic sequences are represented in nuclear RNA approximately once per cell.

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: Characterization of Viral High Molecular Weight Nuclear RNA

SUMMARY High mol. wt. RNA (HMW-RNA) extracted from nuclei of HEp-2 cells 5 h post infection with herpes simplex 1 virus has been shown to have the following characteristics: (i) the amount of HMW-RNA hybridizing to viral DNA fixed on filters increased with input multiplicity as well as following heat denaturation prior to hybridization; (ii) purified self-annealed HMW-RNA was enriched for viral RNA sequences which hybridized to viral DNA following denaturation; (iii) hybridization of excess unlabelled HMW-RNA which labelled viral DNA fragments followed by isopycnic centrifuging in Cs2SO4 led to the partitioning of a fraction of DNA with HMW-RNA. This DNA self annealed at a faster rate than the parental DNA population from which it was derived indicating that the HMW-RNA contained transcripts derived from symmetric transcription of a fraction of viral DNA; (iv) excess unlabelled, heat-denatured, HMW viral RNA drove 50% of viral DNA into DNA-RNA hybrid in hybridization tests with trace amounts of labelled viral DNA. Analysis of the kinetics of hybridization indicated that HMW-RNA consisted of 2 classes arising from 24 to 26% of viral DNA and differing 5000-fold in molar concentration. Since HMW-RNA contains symmetric viral transcripts which self anneal during the hybridization this is probably a minimal estimate of the amount of viral DNA represented in HMW-RNA.

Heterogeneous nuclear rna from lymphocytes of chronic lymphocytic leukaemia: adenylate-rich and double-stranded regions.

Rapidly labelled high molecular weight nuclear RNA from lymphocytes of chronic lymphocytic leukaemia was analysed for ribonuclease-stable adenylate-rich and double-stranded regions. The polyadenylate content corresponds to 0.4-0.5 percent and the content of double-stranded sequences to 2-4 percent of the total nucleotides. Partial association of polyadenylate segments with double-stranded regions was found by comparative analysis of (3H)-adenosine and (3H)-uridine labelled ribonuclease-stable RNA before and after thermal denaturation. Comparison with normal lymphocytes shows lower proportions of polyadenylate-containing RNA binding to poly(U)-Sepharose in leukaemia cells than in normals. Partial degradation of rapidly labelled high molecular weight RNA was found in leukaemia cases with low white cell counts.

Alterations of high molecular weight nuclear RNA following kidney storage

The integrity of nuclear RNA was determined after storage of mouse kidneys. Intraperitoneal 3H-uridine was used to label (30 min) the heterogeneous high molecular weight RNA and ribosomal precursor RNA. The molecular weight distribution of the RNA was analyzed by polyacrylamide gel electrophoresis. Warm ischemia, 60 min at 37°C, resulted in a decrease in the percentage of labeled RNA in the region greater than 45S and the 45S species. The redistribution of the RNA species was not as striking if the kidney was in Collins' solution rather than saline. Additionally, after ischemia there was an increase in label of 32S, 28S, and 18S RNA species. There was no change in the 36S RNA. Storage at 0° for 48 hr in saline produced lower molecular weight products of degradation but no changes in the distribution of the large molcular weight species. Cold storage in Collins' solution prevented this breakdown of labeled RNA. Alterations of the nuclear RNA represented both normal processing and degradation of RNA.

Effect of camptothecin on simian virus 40 DNA.

THE alkaloid camptothecin has been shown to be a potent, rapidly acting inhibitor of RNA and DNA synthesis in eukaryotic cells1–5. The drug is selectively active against high molecular weight nuclear RNA synthesis2–5, having no effect on mitochondrial6 or 4–5S RNA synthesis2,5. Its effect on RNA synthesis is rapidly reversible1–3,5. In addition, it has been shown that camptothecin induces alkali-labile links in DNA1,7–9 which are rapidly reversed by removal of the drug9. It was suggested that this action on DNA is responsible for the effect of the drug on RNA synthesis5.

Isolation and composition of nuclei and nucleoli.

ABSTRACT The past decade has brought about the evolution of increasingly precise methods for isolation of nuclei and nucleoli and their components including histones, acidic proteins, high molecular weight RNA, low molecular weight RNA and ribonucleoproteins. The available methods and improving methods permit the isolation of individual unique molecular species in high states of purity. Elegant studies have been made on the amino acid sequences of some of the histones and on the nucleotide sequences of some of the species of low molecular weight RNAs uniquely found in the nucleus. In addition, nucleotide sequences are being defined for fragments of high molecular weight nuclear RNA. In studies on the effects of hormones on cells, the roles of repressor and derepressor acidic nuclear proteins are now being defined. The next decade should bring extensive new information regarding nuclear structures and functions and nucleolar components and their structures and functions.

Selective interruption of high molecular weight RNA synthesis in HeLa cells by camptothecin.

THE study of macromolecular metabolism in eukaryotic cells has depended to a large extent on the use of selective inhibitors. Camptothecin is a potent, rapidly acting inhibitor of both DNA and RNA synthesis1–3 which first came to attention as a potential anti-tumour agent4 and which has since been shown to have the remarkable property of inducing breaks in cellular DNA5; it also has unusual effects on RNA synthesis possibly as a result of DNA breakage. We show that the drug causes aberrant synthesis of high molecular weight nuclear RNA, but has a much smaller effect on the labelling of 4S RNA and does not affect 5S RNA synthesis. If the effect on RNA synthesis is due to DNA breakage, the results suggest that the breaks are induced at specific points.

Characterization of the RNA synthesized by Phycomyces blakesleeanus

As a part of a survey of simple eukaryotic organisms for the occurrence of heterogeneous, high molecular weight nuclear RNA ( Darnell, Bacteriol. Rev., 32 (1968) 262), we have investigated the RNA of Phycomyces blakesleeanus. High molecular weight nRNA has not yet been found in protozoans such as Amoeba proteus ( Prescott et al. Exp. Cell Res., 64 (1971) 145), Tetrahymena ( Prescott et al., Exp. Cell Res., 67 (1971) 124, or Euplotes ( E. Gamow and D. M. Prescott, unpublished), and whether it is present in any single-celled eukaryotes remains uncertain. In this study P. blakesleeanus, a single-celled, multinucleated fungus ( Bergman et al., Bacteriol. Rev. 33 (1969) 99), was investigated, and the absence of high molecular weight nRNA and the presence of rRNA precursors is described. Incidentally, we draw attention to a technical problem in the quantitative collection of the 4–5-S RNA when glass filters are used.

Presence of cell and virus specific sequences in the same molecules of nuclear RNA from virus transformed cells.

Hybridization studies show that nuclear RNA in transformed cells contains both host and virus specific sequences. This suggests virus-specific mRNA could be formed by selective cleavage of high molecular weight nuclear RNA.