Rates of synthesis and turnover of 5′ cap structures of hnRNA and mRNA and their changes during sea urchin development

The relationship between heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA) synthesis has been studied as a function of the development of the sea urchin embryo through the use of methyl incorporation. Several parameters in the metabolism of capped hnRNA and mRNA of early blastula and late gastrula stages have been investigated by measuring the kinetics of transfer of methyl groups from S-adenosylmethionine to the 5′ cap structures in nuclear and cytoplasmic RNA:

• 1 The rate constants for the decay of hnRNA caps and the synthesis of mRNA caps are equal to within experimental error. This equality indicates a flux of precursor hnRNA caps to mRNA caps with a very high degree of conservation of the hnRNA caps. This conservation holds for each embryonic stage.
• 2 From literature data on the labeling kinetics of GTP and mRNA, we have calculated the decay constant of a putative mRNA precursor component of hnRNA. The value of this constant is very close to that for the decay constant of hnRNA caps. Hence, all hnRNA caps and some portion of their associated hnRNA sequences behave kinetically as the pre-mRNA fraction. This kinetically ascribed pre-mRNA comprises approximately 30% of the hnRNA mass.
• 3 The part of the hnRNA which does not serve as precursor to mRNA turns over at least twice as rapidly as the pre-mRNA fraction.
• 4 During development from early blastula to late gastrula, the rate of hnRNA cap synthesis drops from 2 × 10³ molecules/min/cell to half of this value. This decline is parallel to the decline in total hnRNA synthesis and thereby confirms the constant degree of capping of hnRNA, as previously reported. We infer that the pre-mRNA fraction of hnRNA remains nearly constant during this developmental period.

     

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.     

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.     

Characterization of uterine heterogeneous nuclear ribonucleic acid and the effect of oestradiol-17 beta on its synthesis.

An early response to the administration of oestradiol-17 beta to immature rats is a dramatic stimulation in the synthesis of uterine hnRNA (heterogenous nuclear RNA). High-molecular-weight fractions of the hnRNA were purified and subfractionated on poly(U)-Sepharose into fractions that differed in their poly(A) content and their size profile on polyacrylamide gels. Oestrogen treatment of the rats stimulated the synthesis of all three fractions of high-molecular-weight hnRNA, but the kinetics of synthesis, degree of stimulation and size distribution of the newly synthesize RNA differed in each fraction.     

Effects of N-trifluoroacetyl adriamycin-14-valerate (AD32) on nuclear RNA synthesis in cultured L1210 cells

N-Trifluoroacetyl adriamycin-14-valerate (AD32), an analog of adriamycin, inhibits nuclear RNA synthesis. It inhibits rRNA, non-poly(A) hnRNA and poly(A) hnRNA with essentially no effect on smaller nuclear RNA species (<5S).     

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     

hnRNA and its attachment to a nuclear protein matrix

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.     

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.     

Isolation and characterization of hnRNA-snRNA-protein complexes from Morris hepatoma cells

Of the RNA labelled after incubation of hepatoma cells with radioactive precursors for 20 and 150 min. 35% and 70%, respectively, can be isolated from nuclei by two consecutive extractions with 0.14 M NaCl at pH 8. The isolated RNA is complexed with nuclear proteins forming structures with sedimentation coefficients of less than 30 S to greater than 100 S. Similar complexes from rat liver isolated under the same experimental conditions show coefficients of 30-40 S. The RNA-associated proteins are similar, on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis, to the respective proteins of other cell types. The presence on these RNP complexes of six discrete small nuclear RNAs (snRNA) has been established. Experiments with a reversible inhibitor of RNA synthesis, D-galactosamine, demonstrated, differences in the turnover of hnRNA and snRNA. The half-lives of the six snRNA species has been determined, varying from 32 h for snRNA species a, b and d, to 22 h for snRNA species e and f and to 13 h for snRNA species c. Treatment of the nuclear extracts with 0.7 M and 1 M NaCl results in dissociation of hnRNA from the 'core' and other polypeptides, whereas snRNA remains complexed with polypeptides of Mr 54 000-59 000. Incubation of the nuclear extracts at 0 C with low doses of pancreatic R Nase (up to 1.5 micrograms/ml), which renders approximately 80% of the hnRNA acid-soluble and cleaves most of the snRNA, results in conversion of the high-molecular-weight hnRNPs to 30-S structures, without disrupting the 30-S RNP. Treatment of the nuclear extracts with higher doses of RNase (3 micrograms/ml) leads to disruption of the 30-S RNP and release of the hnRNA-associated proteins, underlining the importance of hnRNA-protein interaction for the retainment of the hnRNP structures.     

Indices of RNA metabolism in the brain stem of rats under superlethal exposure to ionizing radiation

A study was made of the content of hnRNA, nuclear poly(A) RNA and biosynthesis of rlnRNA in truncus cerebri of rats divided into 3 groups by the forced swimming test 6-8 min and 60 min after a short-term exposure to sparsely ionizing radiation of 100 Gy. The observed changes in the nuclear RNA metabolism can subsequently lead to the impairment of the synthesis of proteins required for normal functioning of CNS, and to the development of CNS syndrome.     

hnRNP particles

This article describes the discovery of nuclear DNA-like RNA (dRNA or hnRNA) and ribonucleoprotein particles in eukaryotes. Native hnRNA particles were isolated by sucrose gradient sedimentation and their structural organisation – nucleic acid (i.e. RNA) wrapped in a regular way on the surface of a series of globular protein particles – was determined. This led to the formulation of the informofer cycle hypothesis for the synthesis of hnRNA as a giant precursor molecule, its transport in informosomes within the nucleus, and subsequent splicing before export from the nucleus as free mRNA.     

Preparation in undenatured form of the main protein bound to heterogeneous nuclear RNA in liver and hepatoma cells

Particles carrying heterogeneous nuclear RNA (30 S-particles) were prepared from rat liver and Zajdela hepatoma ascites cell nuclei after ultrasonic disruption. The ribonucleoprotein structures were disintegrated in the presence of 100 mM spermidine. Using chromatography on Sepharose-polyadenylate a protein component has been obtained which possessed high affinity for heterogeneous nuclear RNA, polyuridylate and polyadenylate, and double-stranded DNA. This protein was the main species of the ribonucleoprotein studied; it showed bands with molcular weights of 37000 and 40000 respectively in SDS gel electrophoresis. The RNA-binding proteins isolated from liver and hepatoma had identical molecular weights and the same affinity for Sepharose-polyadenylate used in the isolation.Abbreviations hnRNA heterogeneous nuclear RNA - hnRNP ribonucleoprotein which contains hnRNA     

On the nature of nuclear envelope-associated RNA

Nuclear envelopes were isolated from rat-liver nuclei. Nuclear envelope-associated RNA was isolated and hybridized to filter-bound DNA in the presence of competing RNA populations. Cytoplasmic RNA did not effectively compete for DNA binding sites, while nuclear RNA did. The results indicate a high degree of complexity for nuclear envelope-associated RNA, and are compatible with the idea that hnRNA may be processed after attachment to the nuclear envelope (or nuclear matrix).     

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.     

Delayed processing/export of messenger RNA under conditions of reduced protein synthesis

The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorporation of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acid-starved cells.