A characterization of globin mRNA sequences in the nucleus of duck immature red blood cells

Studies were performed with duck immature red blood cells to identify and characterize the globin mRNA sequences in nuclear RNA. Annealing of ³H-globin cDNA to unlabeled nuclear RNA has identified three distinct size classes of nuclear RNA molecules containing globin mRNA sequences. The largest size class contained 1–2% of total nuclear globin mRNA sequences and sedimented through 85% formamide-sucrose gradients at the same rate as 28S ribosomal RNA. Chromatography on oligo(dT)-cellulose indicated that most of these molecules are not polyadenylated. The bulk of nuclear globin mRNA sequences (70%) was contained in polyadenylated RNA molecules which sedimented at 16.5S. The remainder of nuclear globin mRNA sequences (～30%) was detected in molecules sedimenting at 10S (the position of cytoplasmic globin mRNA).To determine whether a precursor-product relationship exists between these nuclear molecules and cytoplasmic globin mRNA, pulse-label and chase experiments were performed. Labeled globin mRNA sequences were assayed by annealing to globin cDNA-cellulose. Labeled 28S nuclear globin RNA sequences could not be detected, perhaps due to technical reasons. 16.5S nuclear globin RNA was labeled and chased into cytoplasmic globin mRNA sequences. The half-life of 16.5S nuclear globin RNA was estimated to be less than 30 min. These results demonstrate that in duck immature red blood cells, globin mRNA is transcribed as a larger precursor. Furthermore, size characterization of this precursor during pulse-label and chase periods suggests that it is processed within the nucleus to 10S globin RNA.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a large precursor of vitellogenin mRNA in the liver of estradiol-treated chicks.

Studies were performed to determine whether vitellogenin mRNA from avian liver has a precursor molecule or not. Total cellular RNA was prepared from estradiol-treated chicken liver in the presence of 8 M guanidine HCl, 2-mercaptoethanol and aurintricarboxylic acid. After denaturation, RNA was fractionated on sodium dodecylsulfate-sucrose gradients and large size RNA was analyzed under stringent conditions on 85% formamide-sucrose gradients at 25 degrees C. RNA fractions collected from the gradients were hybridized with vitellogenin (3H)-cDNA. Besides mature vitellogenin mRNA (32S, 7,000 nucleotides) vitellogenin sequences were also found in RNA fractions ranging from 38-50S with a peak at 45-50S (12-15,000 nucleotides). Only 5-10% of the putative 38-50S pmRNA is polyadenylated. We calculated that the half-life of vitellogenin pmRNA is about 3-4 minutes. We conclude that vitellogenin mRNA has a precursor which is twice the size of the mature mRNA.     

Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Changes in nuclear and polysomal polyadenylated RNA sequences during rat-liver regeneration.

Nuclear and polysomal polyadenylated RNA populations of normal and 16 hour regenerating rat liver have been compared by mRNA-cDNA hybridisations and by unique DNA saturation experiments. It was found that nuclear polyadenylated RNA hybridises to 6.8% of unique DNA in both normal and 16 hour regenerating rat liver. However, cross-hybridisation experiments using cDNA have shown that 10-15% by weight of nuclear polyadenylated RNA sequences are specific to 16 hour regenerating rat-liver. Since both unique DNA and cDNA hybridisation have shown that normal and 16 hour regenerating rat-liver polysomal polyadenylated RNA populations are qualitatively very similar sequences specific to 16 hour regenerating rat-liver nuclear polyadenylated RNA are nucleus confined. Polysomal RNA sequences which were abundant in normal rat-liver have become less abundant in regenerating rat liver.     

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.     

Relationship between single-stranded DNA isolated from cultured muscular cells during differentiation and the transcription of messenger RNA.

Single-stranded DNA (ssDNA), equivalent to about 2% of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes of the L6 line. Small quantities of 125I-labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non-repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75%) consist of non-self-reassociating DNA sequences and the minor fraction (25%) consists of self-reassociating DNA sequences. About 30--32% and 25--26% of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during mascular differentiation. These differences were confirmed by DNA-driven reactions between 125I-labelled polyadenylated RNA and ssDNA in great excess.     

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.     

Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.     

Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell.     

Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA.80–90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70–80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences.The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.