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.     

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells

Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal — that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.     

Extraction and quantitative precipitation of nuclear ribonucleoprotein particles

Ribonucleoprotein particles (RNPs) were extracted from monkey cell nuclei in media of low ionic strength. The rapidly labeled RNPs were comparable in terms of size, protein patterns and protein content to those extracted by sonication. The overnight labeled RNPs were homogenous (sedimenting at 60-65S and containing RNA of 30-31S), and appear to be a subset of pre-ribosomal RNPs. This procedure produces nuclear RNPs free of contaminating chromatin. Nuclear RNPs (rapidly-labeled and overnight-labeled RNPs, extracted by either our procedure or by sonication), quantitatively precipitated in 10 mM MgCl2 when the concentration of monovalent cations was low. There was no detectable degradation of the RNA components, nor was there loss of enzymatic activity of an RNP associated protein kinase. Precipitation in Mg++ provides a rapid, gentle and convenient method of concentrating RNPs.     

Persistence of nonribosome bound 5 S RNA in full-grown oocytes of Xenopus laevis

The 4 and 5 S RNA containing 42 S ribonucleoprotein (RNP) particles characteristic of previtellogenic and white oocytes cannot be detected in full-grown oocytes. When full-grown oocyte RNPs are separated on sucrose gradients 4 and 5 S RNA cannot be detected in the 42 S region. However, not all of the 5 S RNA stored during early oogenesis is incorporated into ribosomes at later stages. A substantial pool (20% of the total) of 5 S RNA remains in a non-ribosome-bound fraction sedimenting at about 7 S in full-grown oocytes.     

Proteins associated with heterogeneous nuclear RNA in eukaryotic cells

When HeLa cell nuclei axe mechanically disrupted in either hypotonic or isotonic buffers, heterogeneous nuclear RNA is recovered from the post-nucleolar fraction in the form of EDTA-resistant ribonucleoprotein particles, which sediment between 40 S and 250 S in sucrose gradients containing 0.01 m or 0.15 m-NaCl. That the RNA in these particles is HnRNA² is indicated by its heterodisperse sedimentation (20 to 80 S) and its continued synthesis in concentrations of actinomycin D that selectively inhibit the synthesis of ribosomal RNA. The specificity of the HnRNA-protein complexes is evidenced by the failure of deliberate attempts to generate artificial RNP by the addition of deproteinized HnRNA to intact or disrupted nuclei at low ionic strength.The proteins bound to HnRNA are complex. In HeLa cells, HnRNP particles contain proteins with molecular weights from 39,000 to approximately 180,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points between 4.9 and 8.3 (analytical isoelectric focusing). They are readily distinguishable from proteins in other cell fractions, including those in chromatin.Exposure of HeLa HnRNP particles to 0.5 m-NaCl reduces their average sedimentation velocity by approximately 30%. CsCl density-gradient analysis reveals that this is accompanied by the loss of a major portion of the proteins. However, a significant fraction of the HnRNP (25 to 30%) is resistant to high salt concentrations and continues to band at the same density as native HnRNP (1.43 g/cm³). This is true even after prolonged exposure (24 h) to high salt. The salt-resistant HnRNP is enriched for proteins above 60,000 molecular weight. In at least these two respects, this sub-class of HnRNP resembles “messenger RNP” prepared from cytoplasmic polyribosomes, which is also salt-stable and contains relatively high molecular weight proteins.HnRNP particles can also be recovered from HeLa cell nuclei lysed in high salt but these contain many extra proteins, notably histones, and sediment much faster in sucrose gradients than particles prepared as above. HnRNP is not liberated by extracting HeLa nuclei in 0.14 m-NaCl, pH 8.0 (Samarina et al., 1967) unless the temperature is 20 °C or higher. In this case the particles are converted to 45 S structures, which contain partially degraded HnRNA. 45 S particles can also be produced by subjecting 40 to 250 S HnRNP to a very limited digestion with pancreatic ribonuclease (1 to 2 hits/molecule).HnRNP particles have similar sedimentation velocities (40 to 300 S) when isolated under physiological ionic conditions from a variety of mammalian cells, including WI38 human diploid fibroblasts, mouse L-cells, monkey kidney cells and rat liver. However, electrophoresis reveals a distinct pattern of HnRNP proteins for each cell type. It is proposed that this cell-specificity reflects a situation in which HnRNA molecules that differ in nucleotide sequence are complexed with different sets of proteins, so that the resulting HnRNP particles are biochemically distinct at each genetic locus. This hypothesis is discussed in relation to the cytology of lampbrush and polytene chromosomes.     

Ribonucleoprotein particles containing non-coding Y RNAs, Ro60, La and nucleolin are not required for Y RNA function in DNA replication

Background

Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.

Methodology/Principal Findings

We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.

Conclusions/Significance

We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.     

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

Efficient RNA 2'-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C'/D' RNPs

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.     

Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei

Messenger RNA maturation in trypanosomes involves an RNA trans-splicing reaction in which a 39 nucleotide 5'-spliced leader (SL), derived from an independently transcribed 139 nucleotide SL RNA, is joined to pre-mRNAs. Trans-splicing intermediates are structurally consistent with a mechanism of SL addition which is similar to that of cis-splicing of nuclear pre-mRNAs; homologous components (e.g. the U small nuclear RNAs) exist in both cis- and trans-splicing systems, suggesting that these also participate in the two types of splicing reactions. In this study, ribonucleoprotein (RNP) complexes containing the trypanosome SL and U2 RNAs were purified and characterized. Although present at low levels in cellular extracts, the SL and U2 RNPs are the two most abundant of the several non-ribosomal small RNP complexes in these cells. The purification scheme utilizes ion-exchange chromatography, equilibrium density centrifugation, and gel filtration chromatography and reveals that the SL RNP shares biophysical properties with U RNPs of trypanosomes and other eukaryotes; its sedimentation coefficient in sucrose gradients is approximately 10 S, and it is resistant to dissociation during Cs2SO4 equilibrium density centrifugation. Complete separation of the SL and U2 RNPs was achieved by non-denaturing polyacrylamide gel electrophoresis. Proteins purifying with the SL and U2 RNPs were identified by 125I-labeling of tyrosine residues. Four SL RNP proteins with approximate molecular masses of 36, 32, 30, and 27 kDa and one U2 RNP protein of 31 kDa were identified, suggesting that different polypeptides are associated with these two RNAs. These particles are not immunoprecipitated by anti-Sm sera which recognizes U snRNP proteins of other eukaryotes including humans plants and yeast.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Nucleocytoplasmic transport of 5S ribosomal RNA

Nucleocytoplasmic transport of 5S ribosomal RNA in Xenopus oocytes occurs in the context of small, non-ribosomal RNPs. The complex with the zinc finger protein TFIIIA (7S RNP) is exported from the nucleus and stored in the cytoplasm, whereas the complex with the ribosomal protein L5 (5S RNP) shuttles between the nucleus and the cytoplasm. Nuclear import- and export-signals appear to reside within the protein moiety of these RNPs. Import of TFIIIA is inhibited by RNA binding, whereas nuclear transfer of L5 is not influenced by RNA binding. We propose that the export capacity of both, TFIIIA and L5, is regulated by the interaction with 5S ribosomal RNA.     

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix

Summary Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pl of 8.7–8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.Abbreviations CRLM polyclonal chicken antibody raised against rat liver nuclear matrix - Sm monoclonal antibody specific for the 28 kd protein antigen of U1, U2, U4, U5 and U6 snRNPs - hnRNP ribonucleoprotein particles containing hnRNA - snRNP ribonucleoprotein particles containing snRNA - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - VRC vanadium ribonucleoside complex - BSA bovine serum albumin - DMSO dimethylsulfoxide - HS high salt buffer - LS low salt buffer