Core filaments of the nuclear matrix.

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts.

Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA-binding domain. The L protein, therefore, is a new type of hnRNP protein.

Determination of the folding topology of the SL1 RNA from Caenorhabditis elegans by multidimensional heteronuclear NMR.

The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In this study, the first stem loop of the SL1 RNA from the nematode Caenorhabditis elegans was examined by homonuclear and heteronuclear NMR. Results of enzymatic cleavage patterns established that the first 36 nucleotides (which includes the splice site and a complementary base-paired region surrounding a nine-nucleotide hairpin loop) remain structurally independent of the rest of the 100-nucleotide full-length transcript. A comparison of exchangeable and non-exchangeable proton chemical shifts in the region of the splice site and loop between the native sequence and a modified 26-nucleotide fragment from which an asymmetric internal loop had been deleted was made. There was no significant difference between the resonance locations of the equivalent protons in the two molecules, establishing that there was no tertiary interaction between the hairpin and internal loops. Full chemical shift assignments of 1H, 13C, and 15N chemical shifts were obtained for the modified fragment by multidimensional homonuclear and heteronuclear NMR spectroscopy. The stem adopts an A-form helix typical of RNA. The A-type helical conformation of the stem appears to continue for the first three nucleotides of the 5' side of the loop, followed by a guanosine residue in a syn conformation about the glycosidic bond. Base stacking is not seen on the 3' side of the loop. There was no evidence for formation of Watson-Crick base-pairs within the loop, but several long distance NOEs indicated cross-loop contacts, indicative of a structured loop. The final loop residues, an adenine which is conserved among all known nematode SL RNA sequences, adopts an extrahelical conformation.

Oligo(A) and double-stranded segments in polyadenylated and non-polyadenylated RNA from cytoplasm and nuclei of chick embryo.

Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.

[Isolation, characterization, and translation of heterogeneous nuclear RNA from plasmacytoma cells]. / Vydelenie, kharakteristika i transliatsiia geterogennoi iadernoi RNK iz kletok plazmotsitomy.

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.

Protected nucleotide sequences in nuclear ribonucleoprotein.

The rapidly labeled nuclear ribonucleic acid in human carcinoma cells which is protected by protein from digestion by staphylococcal nuclease (EC 3.1.4.7) has been investigated. A simple and discrete sequence specificity was not found, but the protected RNA fragments are rich in G + C and were shown by fingerprinting to comprise a nonrandom subset of all heterogeneous nuclear ribonucleic acid (hnRNA) sequences enriched in the sequences AGC, GGC, AGGC, and GAGC. There was no detectable enrichment for dougble-stranded RNA in the protected fraction. These data provide the first evidence that the association of any protein with hnRNA is nonrandom with respect to nucleotide sequence.

[Sequence complexity of transcribed unique DNA sequences in genome of mouse P815 mastocytoma cells (author's transl)]. / Die Sequenzkomplexität transkribierter unique DNA-Sequenzen im Genom von Maus P815 Mastocytoma Zellen.

The sequence complexity of nuclear RNA from mouse liver, mouse spleen and highly malignant P815 mastocytoma was measured by nRNA driven hybridization to unique DNA sequences of P815 cells. The unique DNA sequences represent 63% of the total nuclear DNA of P815 cells and their availibility in hybridization experiments was found to be 76%. Of these sequences 7.8% formed hybrids with nuclear RNA of this cell, about 11.5% with mouse spleen and about 14.5% with mouse liver nuclear RNA. Assuming an asymmetrical transcription, the complexities of these transcripts are 2.8 X 10(8) nucleotides for mouse P815 mastocytomas, 4.3 X 10(8) for mouse spleen and about 5.3 X 10(8) nucleotides for mouse liver. Cellular specifity of the transcribed information was analyzed in additivity experiments, in which unique DNA sequences, not complementary to the nuclear RNA of one cell were annealed to the nuclear RNAs of the two other tissues/cells. In these experiments most of the nuclear RNA sequences of P815 cells were found to be also present in the nucleus of mouse liver and spleen. Only a small portion of the unique DNA sequences of P815 mastocytoma (about 1.2% corresponding to 4.4 X 10(7) nucleotides) was found to be complementary only to P815 mastocytoma nuclear RNA.

[Biosynthesis and processing of heterogeneous nuclear RNA in rat thymocytes]. / Izuchenie biosinteza i sozrevaniia geterogennoi iadernoi RNK v timotsitakh krysy.

Using [14C]adenine as a labeled precursor, the biosynthesis and processing of heterogeneous nuclear RNA (hnRNA) in chromatin and nucleoplasm were studied. It was shown that intraperitoneally injected actinomycin D (50-500 micrograms/100 g of body weight) did not inhibit the biosynthesis of hnRNA and rRNA in rat thymocytes. Besides biosynthesis, the polyadenylation of the bulk of primary transcripts was also localized in the chromatin. rRNA precursors constituted only a small part of the newly synthesized hnRNA molecules. During processing, highly polymeric (28S-55S) poly(A+)-hnRNA fractions passed from chromatin to the nucleoplasm, as a result of which a large number of high molecular weight polyadenylated hnRNA molecular were pooled in the nucleoplasm. Thymocyte hnRNA was tightly bound to nuclear structures and its isolation tributed stringent conditions. The bulk of poly(A+)-hnRNA molecules were degraded without formation of stable intermediates. The pattern of poly(A-)-hnRNA maturation in chromatin and nucleoplasm was identical, i.e., in both compartments the decrease in the size and accumulation of processed 16S-20S RNA molecules was observed. About 6% of newly synthesized poly(A+)-hnRNA was converted into polysomal mRNA. Cytoplasmic poly(A+)-RNA consists of two fractions, i.e., metabolically stable high molecular weight (greater than or equal to 40S) RNA molecules and a less stable 10S-20S RNA.

Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.

Polyadenylylated heterogeneous nuclear RNA [poly(A)-hnRNA] from mouse brain was subjected to electrophoresis in agarose gels containing CH3HgOH, and molecules larger than 8 kilobases or 13 kilobases were recovered. cDNA was then transcribed from polyadenylylated RNA fragments cleaved from these large molecules. The resulting cDNA hybridized almost to completion with poly(A)-mRNA isolated from mouse brain polysomes. From the hybridization kinetics of this cDNA with its template RNA, it was estimated that the sequence complexity of the 3'-proximal sequences (of the same average size as mRNA) of the greater than 8 kilobase poly(A)-hnRNA was about 57,000 kilobases. The sequence complexity of poly(A)-mRNA, estimated from the template-driven hybridization kinetics of its respective cDNA, was about 110,000 kilobases. It is concluded that most, if not all, of the 3'-proximal sequences of large poly(A)-hnRNA molecules are homologous with mRNA in the mouse brain and that at least 40,000 different mRNA sequences (or portions of mRNA sequences) are represented in the 3'-proximal sequences of greater than 8 kilobase poly(A)-hnRNA.

Interaction of the hnRNA of amphibian oocytes with fibril-forming proteins.

A ribonucleoprotein fraction that contains most of the rapidly labelled hnRNA has been isolated from gently ruptured oocytes of Triturus cristatus. This fraction consists of large aggregates of ribonucleoprotein and has a high (30:1) ratio of protein to RNA. The labelled RNA is contained in ribonucleoprotein particles that have a density of 1.27 g/cm3 in Cs2SO4 gradients (1.39 g/cm3 after formaldehyde fixation in CSCl gradients). Evidence is presented that the particles are associated in vivo with a fibrillar protein network. When the ribonucleoprotein aggregates are treated with ribonuclease, high salt concentration and nonionic detergent, a fibrillar protein residue is produced which contains many species of protein but a few that have electrophoretic characteristics that are identical to major ribonucleoprotein particle proteins. Isolated labelled hnRNA has been shown to bind specifically polypeptides of molecular weight 60 000 and 54 000 that are found in both particle and fibril preparations. In binding assays in vitro, these polypeptides are found to interact with mRNA to a lesser extent and not with rRNA. The isolated 60 000-Mr and 54 000-Mr proteins have the dual ability of forming ribonucleoprotein 'particles' with hnRNA and of polymerizing to generate 10-nm fibrillar structures in the absence of RNA. The possible cellular functions of these proteins are discussed.

HnRNP particles from Drosophila melanogaster cells.

The isolation and characterization of HnRNP from cultured Drosophila melanogaster cells is described. HnRNP particles were extracted from the purified nuclei of sonication in the presence of rat liver cytosol RNAse inhibitor. The nuclear extract was centrifuged on a 15-30% sucrose gradient. The main part of the heterogeneous HnRNP material was localized in the 30 to 80S region of the sucrose gradient. According to the results of re-sedimentation studies the monomer particle was 45S. The buoyant density of HnRNP particles from different regions of the sucrose gradient were equal to approximately 1.4. The protein composition of the particles was analyzed by urea-SDS-polyacrylamide gel electrophoresis. There are five main and a few minor bands. Only the main polypeptides have a slightly higher molecular weight than those of the major polypeptides of 30S subparticles from rat liver nuclei. According to electron = microscopic studies the particles are heterogeneous and the average diameter was found to be 24-26 nm both on the basis of negative contrast and platinum-palladium shadowed pictures.

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.

Improved preparation of poly(A) Sepharose and the isolation of oligo(U) containing heterogeneous nuclear RNA from Friend erythroleukemic cells.

When poly(A) sepharose (prepared according to previously published procedures) was stored in aqueous buffer at 4 degrees C for 5 days or longer, it bound nonspecifically a high percentage of the input RNA which could not be eluted with formamide. We have found that treatment with ethanolamine, followed by dehydration with ethanol yielded poly(A) sepharose which was stable for many months and possessed a low degree of nonspecific binding. Chromatography on poly(A) sepharose permitted the specific isolation of that fraction of Friend erythroleukemic cell heterogeneous nuclear RNA (hnRNA) which contained oligo(U) sequences. Approximately 10% of the hnRNA which contained a poly(A) sequence [poly(A+)] also contained an oligo(U) sequence. Interestingly, prior HCHO denaturation of the hnRNA enhanced binding of the poly(A+) oligo(U+) hnRNA to poly(A) sepharose by tenfold. This suggested that the oligo(U) sequence may be in a region with secondary structure, possibly an intramolecular duplex with the 3' poly(A). Friend cell oligo(U) sequences ranged from 20 to 50 nucleotides in length and, thus, were similar to the oligo(U) sequences which heretofore had only been shown to be present in HeLa cell hnRNA. These results established that rodent cell hnRNA contain oligo(U) sequences and demonstrate, for the first time, that hnRNA containing both a poly(A) and an oligo(U) sequence can be separated from other classes of hnRNA. In addition, conditions are presented for the removal of HCHO from nucleic acid.

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA.

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.

Isolation from Friend erythroleukemia cells of an RNase-sensitive nuclear matrix fibril fraction containing hnRNA and snRNA.

Chromatin-depleted nuclei (CDN) were prepared from Friend erythroleukemia cell nuclei by partial digestion with DNase I and extraction of the chromatin by 2 mM EDTA as described in the preceding paper (Long and Ochs, 1983. Biol. Cell 48, 99-108). These structures contained dense networks of matrix fibrils surrounded by distinct laminae but no morphologically distinct residual nucleoli. CDN disrupted by gentle shearing or 1 microgram/ml RNase were fractionated into laminae and matrix fibrils by differential centrifugation. Protein composition of the lamina fraction was dominated by two prominent lamina proteins that were not detectable in the matrix fraction. Mild RNase treatment led to a conversion of the fibrous network to a particulate morphology while mild shearing resulted in an apparently unaltered fibril fraction. The matrix fibril fractions contained hnRNP proteins and the snRNAs. These results suggest that EDTA-prepared CDN may provide a system for studying snRNP-hnRNP interactions and hnRNP processing that is less complex than intact nuclei.

Fractionation of constituents of ribonucleoproteins containing heterogeneous nuclear ribonucleic acid.

A method of fractionation of hnRNP constituents adaptable to large-scale preparation is presented. It is based on differential resistance to salt dissociation of the two classes of units of hnRNP, the 30--50S monoparticles and the heterogeneous complexes. The monoparticle proteins were released from hnRNP by 0.4 M NaCl. They were separated from the salt-resistant RNP corresponding to the heterogeneous complexes in three steps: chromatography on DEAE-cellulose, high-speed centrifugation, and Bio-Gel chromatography. The latter chromatography permitted a first fractionation of monoparticle proteins according to molecular weight. Such fractions may serve for purification of individual proteins of molecular weight below 80 000. After the two first steps, two fractions of salt-resistant RNP were obtained. In addition to heterogeneous RNA up to 30 S, small nuclear RNAs were detected which represented 6% of total RNA. The protein pattern was complex, and no clear-cut segregation of groups of proteins could be observed between the two fractions. They were both highly enriched in phosphoproteins as compared to nomoparticle proteins. In another fraction corresponding to the void volume of Bio-Gel chromatography, one-third of the RNA was small nuclear RNA. It is suggested that this fraction contains snRNP in addition to free proteins of molecular weight above 80 000 and to salt-resistant RNP similar to those described above but of small size.

Properties of a small transcribed poly A sequence in heterogeneous nuclear RNA of HeLa cells.

A class of heterogeneous nuclear RNA (hnRNA) molecules contain an internal transcribed poly A sequence of close to 25 uninterrupted AMP residues. HnRNA molecules containing this sequence are separable from those containing the large 3' terminal poly A sequence on the basis of their differential affinity for oligo dT cellulose. The fact that the transcribed small poly A and the 3' terminal poly A are not found in the same hnRNA molecules even though both are present in similar size classes and that the small poly A is absent from cytoplasmic messenger RNA (mRNA) has led us to propose a scheme for mRNA processing in which the 3' end of the small poly A in hnRNA becomes a priming size for the post-transcriptional addiction of the large poly A.