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PubMed1981

Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells.

A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220-224) were found in these purified snRNPs, while a significant part of U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cytoplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.

Base Sequence↗
PubMed1983

Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing.

Animals↗
PubMed1981

Adenoviral heterogeneous nuclear RNA is associated with host cell proteins.

In this study irradiation of intact cells with 244-nm ultraviolet light was used to cross-link hnRNA to proteins that are closely associated with it. In this way proteins interacting specifically with hnRNA could be identified excluding the possibility of non-specific binding of proteins to the RNA during cell fractionation. In uninfected HeLa cells two polypeptides of 41500 and 43000 molecular weight can be cross-linked very efficiently to hnRNA. Other proteins, with molecular weights of 36000 and 37000, were covalently linked less effectively. Irradiation of adenovirus-infected cells results in the cross-linking of the same polypeptides to hnRNA. It was found, however, that hnRNA from adenovirus-infected cells contains both viral and host transcripts. Both classes of transcripts are quantitatively associated with the nuclear matrix and can be cross-linked by irradiation with equal efficiency. To determine whether both adenoviral and cellular transcripts are associated with the same proteins, the cross-linked adenoviral hnRNA-protein complexes were isolated by preparative hybridization to adenoviral DNA immobilized on Sepharose. The results show that the purified cross-linked adenoviral hnRNA-protein complexes also contain the host 41500-Mr and 43000-Mr proteins as major components. This suggests that in the infected cell adenoviral-specific hnRNA is associated with host proteins and that the structural organization of viral hnRNA-protein complexes in infected cells probably is similar to the organization of host hnRNA in uninfected cells.

Adenoviridae↗
PubMed1973

Production of viral mRNA in adenovirus- transformed cells by the post- transcriptional processing of heterogeneous nuclear RNA containing viral and cell sequences.

Adenovirus 2-transformed cells contain virus-specific sequences which are covalently linked to cell-specific RNA sequences in heterogeneous nuclear RNA (HnRNA) molecules larger than 45S. Virus sequences are identified by hybridization to viral DNA, and the cell sequences are detected by hybridization to cellular DNA under conditions where hybridization only occurs to reiterated sites in cell DNA. Such large composite viral-cell HnRNA molecules presumably arise through the uninterrupted transcription of host sequences and integrated viral DNA. Adenovirus-specific polysomal RNA from these cells sediments as three discrete species at 16, 20, and 26S. These specific classes of viral mRNA do not contain rapidly hybridizing host-specific RNA sequences. Both virus-specific HnRNA and mRNA contain polyadenylic acid sequences since they bind to polyU columns at levels characteristics of other polyA-terminated HnRNA and mRNA. Thus, the discrete species of virus-specific mRNA in adenovirus 2 transformed cells appear to be derived from high-molecular-weight virus-specific HnRNA through a series of post-transcriptional modifications involving polyA addition. Subsequently the HnRNA is cleaved so that the cell-specific RNA sequences that originate from the reiterated sites in cell DNA do not accompany the adenovirus mRNA to the cytoplasm. These events for the adenovirus-specific mRNA appear, therefore, to be similar to the stages in the biogenesis of the majority of mRNA in eukaryotic cells.

Adenoviridae↗
PubMed1980

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Compartmentation with respect to the synthesis of heterogeneous nuclear RNA and precursors to ribosomal RNA.

The incorporation of [14C]orotate and [14C]uridine into UMP residues of hnRNA (heterogeneous nuclear RNA) and pre-rRNA (precursors to rRNA) of Eharlich ascites-tumour cells was compared: orotate was incorporated at a markedly higher rate into hnRNA. On the other hand, the rate of incorporation of uridine into pre-rRTNA was even somewhat higher than into hnRNA. The ratio of specific radioactivities of CMP to UMP residues in pre-rRNA and hnRNA was studied. At all times of labelling this ratio was similar for both RNA species independently of the precursor used. On addition of excess unlabelled uridine, the CMP/UMP labelling ratio in both pre-rRNA and hnRNA rose. However, this increase was much more pronounced with hnRNA. It is concluded that nuclear pyrimidine nucleotide pool for RNA synthesis is compartmentalized. The synthesis of hnRNa is supplied preferentially by the large and the small compartment, respectively. A detailed model for the cellular compartmentation of uridine nucleotide precursors to RNA is proposed.U

Animals↗
PubMed1981

Electron microscopic observations of heterogeneous nuclear RNA of chicken reticulocytes.

Secondary structures of annealed, snapped-back and untreated chicken reticulocyte poly(A)-containing nuclear RNA were investigated by electron microscopy. Three shapes of molecules were observed: 'coat'hanger' shaped, loop-bearing and filamentous molecules. The former two shaped molecules consist of a small and large fold-back or loop plus a short hook or tail, corresponding to the poly(A) segment at the apparent middle of the structure. The third shaped molecule, mainly seen after snap-back treatment, possesses a small fold-back structure at one end only. It is suggested that the small and large fold-backs or loops of the molecule represent the 5'- and 3'-terminal hairpin structures, respectively. Two distinct sizes of molecule, small and large, irrespective of molecule shape, were observed.

Animals↗
PubMedDate not supplied

[Isolation, characterization, and translation of heterogeneous nuclear RNA from plasmacytoma cells].

It was found that during hot phenol fractionation of plasmacytoma MOPC-21 cells, which produce immunoglobulins of gamma 1 chi-type, separation of nuclear and nucleolar RNAs, differing in kinetics of accumulation of radioactive precursors, distribution during gradient centrifugation procedures and in base composition, takes place. D-RNA 63 degrees and D-RNA 85 degrees, which possess similar characteristics with D-RNAs, discovered earlier in other cells were isolated. It was stated, the D-RNA 85 degrees is considerably enriched by 3'-terminal poly(A) sequences in comparison with D-RNA 63 degrees. During translation in cell-free protein synthesising system from Xenopus oocytes, these two fractions of hnRNA induced formation of immunospecific products. D-RNA 85 degrees stimulated synthesis of gamma 1 and chi chains more efficiently, that D-RNA 63 degrees. Polyadenylated and non-adenylated both D-RNAs stimulated synthesis gamma 1 and chi chains of immunoglobulin G1 during translation. This fact indicates the absence of significant differences in the informational content. These results give opportunity to propose the existence of two levels of processing pre-mRNA for immunoglobulin chains.

Animals↗
PubMed1976

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 disociated contamination 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 cytoplasmic sequences are represented in nuclear RNA approximately once per cell.

Cell Nucleus↗
PubMed1978

Size analysis of heterogeneous nuclear RNA in an aqueous gel system.

We have analyzed the size distribution of heterogeneous nuclear RNA (hnRNA) from cultured murine cells in an aqueous gel system consisting of 1.8% polyacrylamide and 0.5% agarose. hnRNA, labeled in vivo with 32P at a low specific activity, was labeled in vitro at its 3'-end with NaB3H4, and fractionated by gel electrophoresis. The ratio of log 32P/3H cpm was determined for each gel fraction and shown to be linearly related to distance migrated. The ratio of 32P/3H cpm was used to predict the molecular weight of 28-S and 18-S RNAs and shown to be within approx. 10% of the known molecular weight. Denaturation of hnRNA with heat or dimethyl sulfoxide gave similar number and weight average molecular weight when analyzed by gel electrophoresis. The same hnRNA analyzed by sucrose gradient velocity sedimentation had a similar number average molecular weight but much higher weight average molecular weight.

Cell Line↗
PubMed1985

Phosphoproteins crosslinked to poly(A) + heterogeneous nuclear RNA after irradiation with ultraviolet light.

Isolated liver nuclei or whole lymph node lymphocytes stimulated with concanavalin A in culture were irradiated with ultraviolet light. The crosslinked structures of poly(A)+ heterogeneous nuclear RNA and protein were purified on oligo(dT)-cellulose after labelling irradiated nuclei in the presence of adenosine 5'-[gamma-32P]triphosphate and analysed by SDS-polyacrylamide gel electrophoresis. The liver and lymphocyte nuclear proteins included about 17-19 species of 35-150 kDa and were shown to produce quite similar electrophoretic band patterns. Two proteins of 110-120 and 40-42 kDa were phosphorylated. Using partial proteolytic digestion the large-size crosslinked phosphoprotein has been identified as the 110 kDa component described previously (Schweiger, A. and Kostka, G. (1984) Biochim. Biophys. Acta 782, 262-268). The 40-42 kDa band was presumably related to the group C species of main proteins associated with heterogeneous nuclear RNA. In crosslinked nuclear structures from rats treated with low doses of alpha-amanitin for 1 h the relative amount of the 110-120 kDa phosphoprotein was reduced while the labelling with [32P]ATP was almost abolished.

Animals↗
PubMed1984

Effect of convulsions of the synthesis of heterogeneous nuclear RNA associated with polyadenylate and oligoadenylate sequences from El mouse brain as a convulsive strain.

Animals from the El (susceptible to seizures) and ddy (nonsusceptible) mouse strains were subjected to vestibular stimulation by tossing. After convulsions in the El mice, both the stimulated El mice and ddY mice were intracranially injected with [14C]- and [3H]adenosine, respectively. In the control experiment, nonstimulated El and ddY mice received radioactive adenosines in the same manner. The rate of incorporation of adenosine into brain nuclear RNA, expressed as a percentage of the 3H; 14C ratio, was reduced to an average of 68% at 15 min after convulsions, then increased and reached a control value at 5 h. This reduction in nuclear RNA synthesis was not due to alteration of the adenosine triphosphate pool. Gel electrophoresis of RNA revealed no obvious differences in the labeling distribution between El and ddY mice, but the synthesis of RNA species larger than 35S in heterogeneous nuclear RNA (HnRNA) was impaired in convulsed El mice. Nuclear resistant segments of HnRNA with both T1 RNase and RNase A, were chromatographed with poly(U)-Sepharose followed by urea-polyacrylamide gel electrophoresis. The ologo(A) and poly(A) segments consisted of 29, 19, and 11, and 203, 135, and 69 nucleotides, respectively. The convulsions of El mice reduced the incorporation of radioactive adenosines into oligo(A) and poly(A) segments, suggesting that they inhibited transcription as well as polyadenylation within HnRNA.

Adenine Nucleotides↗
PubMed1975

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.

Animals↗
PubMed1979

Analysis of the message-sequence content of the pulse-labeled poly(A)+ heterogeneous nuclear RNA from HeLa cells by cDNA-excess hybridizations.

The message-sequence content of pulse-labeled poly(A)+ HeLa heterogenous nuclear RNA (hnRNA) has been examined by hybridizations to an excess of message cDNA. Control experiments show that the message cDNA accurately reflects the sequence distribution of the complex mixture of poly(A)+ messages present in the HeLa cytoplasm. Pulse-labeled poly(A)+ molecules in both the lamina-associated and shnRNA fractions contain message sequences, and approximately 65% of the poly(A)-adjacent hnRNA sequences are homologous to the 3' ends of mRNA. The majority of the pulse-labeled hnRNA molecules contain abundant message sequences. By use of these techniques it is also shown that some pulse-labeled polyadenylated message sequences are still synthesized in the presence of the adenosine analogue 5,6-dichloro-beta-D-ribofuranosylbenzimidazole under conditions where little or no new cytoplasmic mRNA is produced.

Base Sequence↗