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PubMed1977

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.

Animals↗
PubMed1987

Nuclear ribonucleoprotein immune complexes in pericardial fluid of a patient with mixed connective tissue disease.

We performed immunopathologic studies of the pericarditis present in a patient with mixed connective tissue disease. A large number of nuclear RNP (nRNP) immune complexes (ICs) were found in the pericardial fluid, but not in the serum. The pericardial small vessels had no deposits of IgG. These results suggest that locally formed nRNP ICs were closely associated with the pathogenesis of pericarditis in this patient.

Adult↗
PubMed1979

Nuclear ribonucleoprotein complexes of amphibian liver. I. Characterization of the complex and its small molecular weight RNA moiety.

Nuclear RNA-protein complexes containing small molecular weight RNAs were isolated from hepatic nuclei of Rana catesbeiana tadpoles and frogs according to a procedure normally used for the isolation of heterogeneous nuclear ribonucleoprotein complexes from other eukaryotic tissues. Preliminary characterization of the tadpole nuclear RNP indicated a particle size of 50--70 S in sucrose density gradients and a buoyant density of 1.40 gm/ml in CsCl gradients. When analyzed on SDS-polyacrylamide gels, this complex was observed to contain at least 40 polypeptides ranging in molecular weight from 15,000 to 200,000. Nuclear RNA-protein complexes were also isolated from adult frog hepatic nuclei by the same protocol and the RNA moiety which had been purified from the frog complex was compared with the nuclear RNA isolated from the tadpole particles. Electrophoretic analysis of the nuclear RNA-protein-associated RNA revealed minor qualitative and quantitive differences in the more than 25 discrete bands (4--9 S) associated with each particle. Base analysis of tadpole and frog nuclear RNA revealed a nucleotide composition of approximately 50% adenosine plus uridine nucleotides, with an unusually high content of cytosine residues (approximately 30%). Comparison of the two RNA samples demonstrated a large increase in the adenosine content of frog unclear RNA, and the presence of a minor base in frog nuclear RNA which was absent in the tadpole sample. These results indicated that changes in the RNA content of the amphibian nuclear RNP complex had occurred during bullfrog development.

Animals↗
PubMed1981

Analysis of nuclear proteins in primary spermatocytes of Drosophila hydei: The correlation of nuclear proteins with the function of the Y chromosomal loops.

The protein content of spermatocyte nuclei from X/Y males and mutants of D. hydei which lack different Y chromosomal loop forming sites, was compared with that of X/0 males in 14C/3H double labelling experiments. Proteins of 45,000, 52,000, 54,000, 66,000, 80,000, 84,000 and 170,000 Dalton are found to be enriched in nuclei containing two or more active Y chromosomal loop forming sites. These proteins are also present in the nuclei of X0 males. In the complete absence of the Y-chromosomal loops proteins of 35,000, 46,000, 58,000 and 110,000 Dalton become enriched in the spermatocyte nuclei. - Analysis of the nuclear RNP of spermatocytes led to the isolation of an hnRNP-containing fraction with an S-value of greater than 900S (RNP-PP), - In the RNP-PP of XY males labelled protein material associated with hnRNA is enriched by a factor of approximately 3 in respect to the X0 genotype. The nuclear RNP has a heterogenous buoyant density in CsCl of rho = 1.33 to 1.43 g/cm3. RNase T1 treatment of the crude nuclear RNP from XY males prior to sucrose gradient analysis shows that the 66,000 Dalton protein which is also strongly enriched in the nuclei in the presence of active Y chromosomal loop forming sites, is the main protein associated with protected RNA-sequences of 80-120- 300 nucleotides in length. Competitive nitrocellulose filter binding assays reveal that the 66,000 Dalton protein predominantly forms in 2 M NaCl stable RNA/protein complexes with the poly A+hnRNA of the RNP-PP. Those RNP complexes have a buoyant density of rho = 1.43 g/cm3 in CsCl. The results are discussed in relation to the nuclear structure and the function of the Y chromosomal loops during spermatogenesis in Drosophila hydei.

Animals↗
PubMed1983

In vitro reconstitution of 35S ribonucleoprotein complexes.

Ribonucleoprotein complexes (hnRNP) sedimenting at 30-40 S and containing fragments of heterogeneous nuclear RNA (hnRNA) have been extracted from HeLa cell nuclei. Besides hnRNA fragments (8-12 S), the complexes contain eight mostly basic core proteins of Mr 31 000-41 000 as shown by two-dimensional gel electrophoresis. Other proteins (mostly of higher molecular weight) seem to be peripherally associated since they are lost after pelleting and recentrifugation of the hnRNP complexes. The particle dissociates into its protein components after digestion of the endogenous hnRNA fragments by micrococcal nuclease. After inactivation of the nuclease and addition of a wide variety of exogenous RNAs [MS2 phage RNA, poly(U), poly(C), poly(A), and poly(A,U)], a RNP particle is re-formed which resembles the native hnRNP complex according to its sedimentation value (35 S), its appearance in the electron microscope, its density in metrizamide, and its protein composition. No particles are formed on double-stranded RNA [poly(A) . poly(U)] or native DNA whereas denatured DNA allows complex formation. On MS2 RNA (3569 nucleotides), the formation of tri- and tetrameric complexes is observed. This indicates the presence of 900-1200 nucleotides per particle. In vivo, 40S hnRNP particles are a unit component of larger RNP structures. Hence, we conclude from our results that the hnRNP core proteins have the intrinsic capability to associate with nascent single-stranded hnRNA regions to form these RNP complexes. Because of the lack of any sequence specificity, the complexes may function in packaging of the hnRNA and in connection with other nuclear components may provide a scaffold for subsequent processing reactions.

Cell Nucleus↗
PubMed2004

Ylidene-->iminophosphine coordination complexes and reversible dissociation of dichlorophosphetidines.

Chloro-, bromo-, iodo-, and trifluoromethylsulfonyloxy-(2,4,6-tri-tert-butylphenylimino)phosphines (MesNPX; X = Cl, Br, I, OTf) react quantitatively with 1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene (Im) to give Lewis acid-base complexes with the general formula MesNP(Im)X. The dichlorophosphetidine (DippNPCl)(2) (Dipp = 2,6-diisopropylphenyl) represents a formal cyclodimer of an iminophosphine and reacts with Im to give a similar complex. The process represents a ligand induced dissociation of the phosphetidine framework and is reversed by the introduction of an appropriate Lewis acid. Solid state structures of RNP(Im)X complexes show that the closest contact between acid and base occurs between phosphorus and carbon in all cases, highlighting them as compounds that contain examples of C-->P coordinate bonds. Association of Im with phosphorus also effects a substantial increase in the P-X distance, but all derivatives maintain a short NP bond, indicating the presence of NP pi-bonding.

Journal Article↗
PubMed2003

G-quartet-dependent recognition between the FMRP RGG box and RNA.

Fragile-X syndrome, the most common monogenic form of mental retardation, is caused by down-regulation of the expression of Fragile X Mental Retardation Protein (FMRP). FMRP is a multifunctional, multidomain RNA-binding protein that acts as a translational repressor in neuronal cells. Interaction between FMRP and mRNA targets involves an RGG box, a protein motif commonly thought to mediate unspecific interactions with nucleic acids. Instead, FMRP RGG box has been shown to recognize RNA G-quartet structures specifically and to be necessary in neurons for RNP particle formation and dendritic mRNA localization. In the present study, we have characterized structurally three representative RNA targets of FMRP in their unbound form and in complex with the RGG box. We observe a large heterogeneity in the conformation of the RNA targets and in their RGG binding mode, which could be the basis of recognition specificity. We also found that G-quartet formation occurs not only intramolecularly but can also be mediated by RNA dimerization. These findings suggest a potential role of RNA:RNA interactions in protein:RNA complexes and in RNP particle assembly.

Amino Acid Sequence↗
PubMed1984

Characterization of the autoimmune antigenic determinant for ribonucleoprotein (RNP) antibody.

Small nuclear ribonucleoprotein complexes are antigens in various autoimmune diseases. The serological pattern of high titers of circulating antibody to nuclear ribonucleoprotein (RNP) antigen is a diagnostic marker for mixed connective tissue disease (MCTD); whereas antibody to Sm is prevalent in systemic lupus erythematosus (SLE). Both calf thymus and rabbit thymus are commonly used, excellent sources for preparation of the corresponding antigens RNP and Sm in clinical and research laboratories (A. M. Boak et al., accompanying paper). Thus, biochemical and structural characterization of the minimal antigenic determinant in these preparations is important for its use in the laboratory, as well as significant for understanding MCTD, SLE, and other examples of autoimmunity. Purification and biochemical analyses of immunologically active RNP from many different preparations of calf thymus extract has revealed that the majority of antibody in monospecific MCTD patient sera recognizes an antigen composed of the 165 nucleotide RNA, U1 RNA, and five peptides. Calf thymus U1 RNA was found to be identical in sequence to that of man. A sequence of 55 nucleotides within the 165 nucleotide RNA was the minimal RNA fragment found in RNP particles that were still immunologically active. Two of the RNP peptides react with patient sera monospecific for RNP and thus, are presumably the antigenic peptides complexed with the 55 nucleotide RNA sequence.

Animals↗
PubMed2001

Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA.

Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike alpha-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) alpha complex on its 3' untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human beta-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3' UTR of the beta-globin mRNA that exhibits many of the properties of the stability-enhancing alpha complex. The beta-globin mRNP complex is shown to contain one or more factors homologous to alphaCP, a 39-kDa RNA-binding protein that is integral to alpha-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3' UTR as the site of beta-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human beta-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the alpha- and beta-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.

3' Untranslated Regions↗
PubMed1996

Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).

Base Sequence↗
PubMed1998

Initiation of hepatitis delta virus genome replication.

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (deltaAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant deltaAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input deltaAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of deltaAg-S. It is this second source of deltaAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for deltaAg-S with truncated or modified forms of the deltaAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of deltaAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

Amino Acid Sequence↗
PubMed1985

Detection of beta-globin mRNA precursor in heterogeneous nuclear ribonucleoprotein associated with U1-RNP by using anti-RNP antibody.

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.

Animals↗
PubMed1976

Morphology of transcription units in Drosophila melanogaster.

We have used an electron microscopic analysis to define and to characterize active transcription units of Drosophila melanogaster. The lengths and spacings of nascent ribonuclear protein (RNP) fibers were determined on embryonic chromatin that was spread using techniques introduced by Miller and Beatty (1969). The data are consistent with the occurrence of specific sites of transcription initiation and termination. We apply the term transcription unit (TU) to a chromatin region bounded by these control sites. Two classes of TUs are active in Drosophila melanogaster embryonic cells--those synthesizing ribosomal RNA and those synthesizing non-ribosomal RNA. The classes can usually be distinguished on the basis of TU size, chromatin morphology and inferred DNA packing ratio, frequency of RNP fibers (number of fibers per mum of chromatin), and the solitary vs. tandem repeat occurrence of fiber arrays. The results indicate that non-ribosomal transcription units have lengths in accord with the expectation that DNA of each chromomere is transcribed as a unit. Some nascent fiber arrays in D. melanogaster have more complex patterns of RNP fiber lengths. We suggest that these are a consequence of cleavage of RNP fibers at specific sites during transcription. These sites of transcriptional control and the amounts of DNA between them provide a basis for further relating units of transcription to units of gene function.

Animals↗
PubMed2002

Differentiation of RNP- and SM-antibody subsets in SLE and MCTD patients by a new ELISA using recombinant antigens.

Connective tissue diseases often have overlapping clinical features and laboratory abnormalities. The distinctiveness of mixed connective tissue disease (MCTD) as an entity is of scientific interest and practical importance. In order to discriminate between MCTD and SLE patients we used a newly developed, commercially not available ELISA with recombinant antigen expressed in Baculovirus infected cells. This ELISA detects antibodies against RNP and Sm in complex as well as the subsets U1-snRNP 68 kDa, RNP-A, RNP-C (RNP), Sm-BB' and SS-D. We analyzed 66 RNP-positive consecutive patients prediagnosed as SLE or MCTD/overlap-syndrome. 45/66 patients were found to be U1-snRNP-68 kDa positive (27 SLE, 18 MCTD), 51/66 RNP-A [36,15] and 44/66 RNP-C [31,13]. 35/66 had antibodies against Sm-BB' (30 SLE, 5 MCTD), 10/66 against Sm-D (all SLE). 28/66 were found to be U1-snRNP-68 kDa and Sm-BB' positive (23 SLE, 5 MCTD), while 8/66 where U1-snRNP-68 kDa and Sm-D positive (all SLE). The combination of antibodies against 68 kDa, Aand C was exclusively observed in 6 MCTD patients, while the combination against 68 kDa, A, C, Sm-BB' and Sm-D was restricted to 8 patients with SLE. The antibody combination to 68 kDa, A, C and Sm-BB' was also found in 11/20 SLE patients with major organ involvement. In SLE and MCTD, determination of subsets of antibodies against Ul-snRNP-68 kDa and Sm-complex allows a differentiation of patient subgroups with more definite diagnoses and potential prognostic impact.

Adult↗
PubMed1987

The autoimmune antigen Me is distinct and related to undifferentiated connective tissue disease.

Using prototype Me serum, a new autoantibody-antigen system has been identified by Ouchterlony immunodiffusion and indirect immunofluorescence. Although immunologically distinct, the Me antigen has physiochemical and biochemical properties similar to those of the Sm antigen. Immunoblot assays indicate that Me sera commonly recognize 4 peptides of molecular weights approximating 100K, 65K, 21K, and 16K. The last of these may be identical to the D peptide recognized by Sm antibodies. The Me antigen may be associated with the RNP-Sm macromolecular complex. Me-positive patients have few clinical symptoms, and the most common diagnosis is undifferentiated connective tissue disease.

Antigen-Antibody Complex↗
PubMed1982

Age-dependent gene induction in quail oviduct. XV. Alterations of the poly(A)-associated protein pattern and of the poly(A) chain length of mRNA.

The effect of ageing on polyadenylate [poly(A)] metabolism of mRNA was studied in two age groups of female quails: mature (250-320 days' old) and senescent animals (3-3.5 years' old). In introductory experiments it was shown that poly(A)-associated proteins can not be recovered from cytosol by affinity chromatography. We isolated the poly(A)-associated proteins from polyribosomal poly(A)-ribonucleoprotein complex [poly(A)-RNP] and radioactively labeled them with dansyl chloride. Three main protein species were identified with molecular masses of 48000 (P48), 35000 (P35) and 24000 (P24). During ageing the percentage portion of P48 in poly(A)-RNP from liver (mitotic tissue) and from oviduct or heart (post-mitotic tissue) is reduced at the expense of P35 and P24. Quantitative analyses revealed that the amount of poly(A)-RNP in the different organs decreases significantly with age if the values are based on DNA. The protein content in poly(A)-RNP was found to be reduced especially in post-mitotic tissue. From this finding we assume that the number of poly(A)-associated protein molecules per poly(A) stretch drops from approximately 4.7 molecules (mature oviduct) to 1.9 molecules (senescent oviduct). Control experiments revealed that free, non-polyribosomal poly(A)-RNP accounts only for 10% of total poly(A)-RNP. The size of the poly(A) segment of mRNA decreases with age. After labeling with [3H] dimethylsulfate, the poly(A) stretch from mature oviduct was found to consist mainly of 120-180 AMP units, and those from mature liver and mature heart of 110 and 100, respectively. In organs from senescent animals the percentage of shorter poly(A) stretches is enlarged; on the average, poly(A)-70 chains were detected. These results support the assumption that age-dependent changes occur also on the post-transcriptional level during the maturation steps of poly(A)(-) hnRNA to poly(A)-(+) mRNA.

Aging↗
PubMed1982

Assembly of transcriptionally active 5S RNA gene chromatin in vitro.

We have studied the requirements for the in vitro assembly of transcriptionally active 5S RNA gene chromatin from cloned Xenopus laevis 5S plasmid DNA. Both plasmid DNA and DNA assembled into chromatin with Xenopus oocyte extracts are transcribed efficiently in vitro. Chromatin prepared by NaCl reconstitution with purified histones in the absence of any cellular factors, however, is transcriptionally inert. A transcriptionally active template is formed if plasmid DNA is incubated in an ovary extract prior to, but not after, NaCl reconstitution. The cellular component responsible for this effect is the 5S RNA transcription factor TFIIIA. Both chromatographically purified TFIIIA and TFIIIA derived from 7S RNP particles can complex with 5S DNA to yield an active chromatin template upon reconstitution with histones. This effect is specific for 5S RNA genes, since TFIIIA will not form an active template when incubated with a cloned Bombyx mori alanine tRNA gene.

Animals↗