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.

Nucleic-acid-binding properties of hnRNP-U/SAF-A, a nuclear-matrix protein which binds DNA and RNA in vivo and in vitro.

We show that SAF-A, a nuclear protein which specifically binds vertebrate scaffold-attachment-region (SAR) elements with high affinity is identical with hnRNP-U, assumed to be involved in packaging of hnRNA in ribonucleoprotein particles. Ultraviolet cross-linking experiments show that the protein, referred to as hnRNP-U/SAF-A, is bound to chromosomal DNA in vivo. In vitro, the isolated protein binds to double-stranded and single-stranded DNA and forms higher ordered nucleic-acid-protein complexes. Filter-binding experiments performed with different types of natural and synthetic nucleic acids as substrates show that the protein binds DNA and RNA with different affinities and most likely at different binding sites. We conclude that hnRNP-U/SAF-A thus may have functions in the organisation of chromosomal DNA in addition to its suggested role in hnRNA metabolism.

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.

Identification of chromosome-specific sequence-tagged sites by Alu-PCR.

Recently, methods have been developed for the isolation of expressed sequences from particular human chromosomes. Using Alu consensus sequences as primers, cDNA synthesis has been initiated from interspecies hybrid cell lines that contain single human chromosomes. Alu consensus sequences have also been utilized to amplify human genomic sequences via polymerase chain reaction (PCR). Here, we describe the use of Alu-PCR to isolate expressed sequences from human chromosomes selectively. Heteronuclear (hn) RNA is transcribed into cDNA by using poly-(dT)15 primer sequences. Subsequently, human specific cDNA sequences are amplified by Alu-PCR and cloned into pBluescript. To verify the chromosomal assignment, cloned PCR products are sequenced, converted into STS markers, and tested on a different somatic hybrid that contains human chromosome 22. The method provides a fast, reliable way to identify expressed sequence tagged sites from selected human chromosomes.

Cytoskeletal messenger RNA stability in human neocortex: studies in normal aging and in Alzheimer's disease.

Total RNA was extracted from human brain temporal and parietotemporal neocortical grey matter with postmortem intervals (PMI) of up to 13.5 hours. The integrity and rank abundance of heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA) were analyzed by Northern gel dot blot hybridization with specific cloned probes of neurobiological interest: the RNA messages for four cytoskeletal components including glial fibrillary acidic protein (GFAP), alpha-tubulin, beta-actin and the human neurofilament light chain (HNF-L) genomic sequence, the Alu repetitive element, the scrapie prion PrP DNA probe and the chromatin condensing agent linker histone H1(0) genomic probe. Our observations indicate that for the cytoskeletal RNA messages studied here: (1) short postmortem intervals (of up to 4.5 hours) had only small effects upon RNA quality in these neocortices, (2) GFAP and HNF-L transcripts were represented at relatively high levels in the cerebral neocortex and (3) each RNA species in normal human brain had both unique and characteristic intracellular levels of abundance and decay kinetics. In the pathological condition, Alzheimer's disease (AD), cells of the temporal and parietotemporal neocortices of afflicted brains showed selective reductions in cytoskeletal RNA pool size which are not attributable to RNA transcript stability.

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.

[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.

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin- and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.

Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.

A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.

[Relation between the structural, metabolic and "adhesive" properties of nuclear and cytoplasmic non-ribosomal RNA]. / Vzaimosviaz' strukturnykh, metabolicheskikh i "adgezivnykh" svoistv iadernykh i tsitoplazmaticheskikh neribosomnykh RNK.

Nuclear RNAs release from nucleoproteins of isolated nuclei absorbed on a celite column in a wide range of dissociating conditions (from 1 M LiCl--2 M urea at 2 degrees C to 4 M LiCl--8 M urea at 70-80 degrees C) was demonstrated. Such a high "adhesive" heterogeneity of nuclear RNAs (i.e., variations in the tightness of RNA-protein bonds) appears to be due to the association of nuclear matrix proteins. A direct correlation was found to exist between the metabolic turnover of RNA and the tightness of its association with the nuclear matrix. Actually, the pulse label which rapidly incorporates into the RNAt greater than 50 degrees, the RNA fraction being most tenaciously bound to the matrix, could be chased later into RNAs weakly bound to it. As the RNA-matrix binding weakens, the metabolic and structural properties of a given RNA change, e.g., sedimentation coefficients decrease, while the poly(A)+-RNA content and stability increase. The "adhesive" heterogeneity was found to be inherent in not only nuclear RNAs but also in cytoplasmic non-ribosomal RNAs, showing the same correlation, i.e., the tighter the RNA--protein complex, the higher the rate of RNA turnover. Cytoplasmic RNAs which differ in their adhesiveness may fulfil various intracellular functions, since polyribosomal mRNPs and informosomal mRNPs appear to be enriched in tightly and weakly bound RNA fractions, respectively. The interrelationships between nuclear and cytoplasmic RNAs are discussed.

Glucocorticoids decrease the synthesis of type I procollagen mRNAs.

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.

Isolation and characterization of nuclear particles containing rapidly labelled hnRNA and snRNA in combination with a distinct set of polypeptides of Mr 74000 and 72000.

A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) [1]. The RNP sediments in sucrose gradients with s-values of 70-110S. Formaldehyde-fixed preparations band at Q = 1.40 in isopycnic CsCl gradients. The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with Mr 74000 and 72000. It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophoresis. Treatment of rats with alpha-amanitin leads to a significant decrease in the amount of recovered RNP. In the presence of 0.7 M NaCl the s-value of the complex changes from 70-110S to 40-80S. The RNP structure is stable to mild RNase A or micrococcal nuclease digestion. Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300-360 A. The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al. [29].

Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures.

Lytic infection with polyoma virus leads in Go-arrested primary mouse kidney cell cultures to a mitotic host response. In the present work we focused our attention on cellular RNA synthesis shortly after onset of polyoma T-antigen synthesis. Onset of polyoma-induced stimulation of 45S pre-rRNA synthesis was determined by hybridization of total cellular RNA with a plasmid (pMrSalB) containing the 5'-end of the mouse ribosomal gene and of the other cellular RNA species by standard biochemical analysis of cellular fractions. The results showed that polyoma-induced stimulation of cellular hnRNA (hnRNP) synthesis, the earliest presently known host cell reaction, preceded onset of stimulated 45S pre-rRNA synthesis and that the latter was paralleled by polyoma-induced stimulation of 5S RNA, tRNA and overall protein synthesis. The polyoma-induced mitotic response is similar to that triggered by simian virus 40 and by certain nonviral mitogens.

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.

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.

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.

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.