Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single- stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.     

Globin mRNA precursor. Cross-linking in situ of double-stranded segments with aminomethyltrioxalen.

The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.     

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.     

Kinetics of formation of 5′ terminal caps in mRNA

A kinetic analysis of the labeling of the methylated components of messenger RNA and heterogeneous nuclear RNA in mouse L cells indicates that the 5′ terminal cap I structures (m⁷GpppX^mpYp) of mRNA are derived from 5′ terminal cap structures of hnRNA. Most of the hnRNA caps are conserved during processing, whereas only a portion of the internal m⁶A residues in hnRNA are conserved. The cap II structures (m⁷GpppX^mpY^mpZp), which constitute the 5′ termini of some mRNAs, arise by a “secondary” methylation that occurs after the mRNAs have entered the cytoplasm. This secondary methylation is apparently restricted to a particular subclass of mRNAs having a high frequency of pyrimidine nucleotides at position Y, a composition at position X which differs from that of the bulk of the cap I-terminated mRNAs, and a relatively slow rate of turnover.     

Comparative inhibition of nuclear RNA synthesis in cultured mouse leukemia L1210 cells by adriamycin and 4'-epi-adriamycin

Adriamycin and 4'-epi-adriamycin were compared as to their effect on nRNA synthesis. 4'-Epi-adriamycin was a more effective inhibitor than the parent compound of RNA synthesis as measured by incorporation of [3H]-uridine. Adriamycin inhibited all three species of nRNA (ribosomal, non-poly(A)hnRNA, poly(A)hnRNA) to approximately the same extent. 4'-Epi-adriamycin on the other hand inhibited the nRNA species in the following order: non-poly(A)hnRNA greater than ribosomal RNA greater than poly(A)hnRNA. The inhibitory effects of both drugs on incorporation of uridine into RNA were reversible at low concentrations (5 microgram/ml).     

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.     

Pulse and steady-state labelled and actinomycin D chased 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-resistant hnRNA from uninfected HeLa cells

The fraction of hnRNA synthesized in the presence of DRB in uninfected HeLa cells ranges in size from 4S to over 45S. High molecular weight DRB-resistant hnRNA has been demonstrated to decay after 2 h of actinomycin D chase. This fraction is composed of both poly(A)(+) and poly(A)(-) RNA molecules. The synthesis in the presence of DRB of short polyadenylated hnRNA was also observed. The nature of both hnRNA subfractions is discussed.     

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.     

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.     

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