Distribution of Low-Molecular-Weight RNAs in Avian Erythroblast Nuclear Ribonucleoprotein Complexes Associated with Pre-messenger RNA (original) (raw)
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European Journal of Biochemistry, 1979
Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.
Biochimica et biophysica acta, 1979
70-130 S polyparticles as well as 38 S monoparticles were isolated from rat liver nuclei and analyzed in respect to their RNA components by microgel polyacrylamide electrophoresis in formamide. In addition to the high molecular weight polydisperse hnRNA of polyparticles several low molecular weight RNAs (snRNA) were detected. There are at least six distinct snRNA species in polyparticles. Except for one species, which is missing, 38 S monoparticles showed a similar snRNA pattern. From densitometer tracings of microgels the snRNAs were estimated to represent about 11% of the total polyparticle RNA. The number of nucleotides for the various snRNAs were determined from a plot of relative electrophoretic mobility versus log number of nucleotides. The possibility that the snRNAs are degradation products of the hnRNA was excluded on the basis of the following findings. (1) The snRNA pattern was similar in mono- and polyparticles. (2) Whereas the hnRNA of polyparticles incubated at 37 degr...
Small nuclear RNA transcription and ribonucleoprotein assembly in early Xenopus development
The Journal of Cell Biology
The Xenopus egg and embryo, throughout the transcriptionally inactive early cleavage period, were found to contain a store of approximately 8 x 108 molecules of the small nuclear RNA (snRNA) U 1, sufficient for 4,000-8,000 nuclei. In addition, when transcription is activated at the twelfth cleavage (4,000 cell-stage), the snRNAs U1, U2, U4, U5, and U6 are major RNA polymerase II products. From the twelfth cleavage to gastrulation, U1 RNA increases sevenfold in 4 h, paralleling a similar increase in nuclear number. This level of snRNA transcription is much greater than that typical of somatic cells, implying a higher rate of U1 transcription or a greater number of U1 genes active in the embryo. The Xenopus egg also contains snRNP proteins, since it has the capacity to package exogenously added snRNA into immunoprecipitable snRNP particles, which resemble endogenous particles in both sedimentation coefficient and T1 RNase digestibility. SnRNP proteins may recognize conserved secondary structure of U1 snRNA since efficient packaging of both mouse and Drosophila U1 RNAs, differing 30% in sequence, occurs. The Xenopus egg and embryo can be used to pose a number of interesting questions about the transcription, assembly, and function of snRNA.
Electron microscopic studies of purified eukaryote messenger RNA
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1977
Rat liver mRNA, hen and rabbit globin messenger RNA (mRNA) were investigated by electron microscopy. No secondary structures were visible in the molecules of these mRNAs under conditions where short secondary structures of other types of RNA, i.e. MS2 phage RNA and 28 S rRNA, were clearly demonstrated. The contour lengths of these mRNAs were also determined by electron microscopy and compared with the sizes estimated by other techniques. The contour lengths of rabbit short and long globin mRNA are 0.1498 + 0.0019 and 0.1908-+ 0.0021 pm, respectively. The former is assumed to be globin mRNA for a chain and the latter for/3 chain. Similarly, hen "a" and "/3" globin mRNA have mean lengths of 0.1449 + 0.0011 and 0.1891-+ 0.0017 pm, respectively. Hen and rabbit reticulocytes contain 1.8-2.0 times as much mRNA for globin chains as for ft.
Cell, 1996
involved in regulation of gene expression at different and Bertil Daneholt levels. Some of them have been found to bind DNA and Department of Cell and Molecular Biology might act as transcriptional regulators (e.g., Tomonaga Medical Nobel Institute and Levens, 1995). Others are involved in pre-mRNA Karolinska Institutet processing: for example, human hnRNP A1 plays a role S-17177 Stockholm in splice-site selection both in vitro and in vivo (Mayeda Sweden and Krainer, 1992; Cá ceres et al., 1994), and HRB98DE, probably the closest relative of A1 in D. melanogaster, is involved in alternative splicing as well (Shen et al., Summary 1995). Possible roles in mRNA export and essential functions in the cytoplasm have been suggested for some In the larval salivary glands of C. tentans, it is possible of the shuttling hnRNP proteins in mammals and insects to visualize by electron microscopy how Balbiani ring (Piñ ol-Roma and Dreyfuss, 1992; Kelley, 1993; Matunis (BR) pre-mRNA associates with proteins to form preet al., 1994). mRNP particles, how these particles move to and The intracellular location of different hnRNP proteins through the nuclear pore, and how the BR RNA is was analyzed in cultured cells by immunofluorescence engaged in the formation of giant polysomes in the microscopy (reviewed by Piñ ol-Roma and Dreyfuss, cytoplasm. Here, we study C. tentans hrp36, an abun-1993). Initial observations indicated that the major dant protein in the BR particles, and establish that it hnRNP proteins were confined to the cell nucleus, and is similar to the mammalian hnRNP A1. By immunothus it was suggested that hnRNP proteins would dissoelectron microscopy it is demonstrated that hrp36 is ciate from the RNP complexes before or during nucleoadded to BR RNA concomitant with transcription, recytoplasmic transport (reviewed by Dreyfuss, 1986). mains in nucleoplasmic BR particles, and is translo-Data from early biochemical studies (e.g., Kumar and cated through the nuclear pore still associated with Pederson 1975; Martin et al., 1980) also supported the BR RNA. It appears in the giant BR RNA-containing view that pre-mRNA molecules are bound to a set of polysomes, where it remains as an abundant protein proteins in the nucleus (hnRNP proteins) and to a differin spite of ongoing translation. ent set of proteins (mRNP proteins) in the cytoplasm. Indeed, some of the hnRNP proteins characterized so
The Journal of cell biology, 1980
To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with c...
Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles
Proceedings of the National Academy of Sciences, 1987
Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles Ul to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights-150,000 and 115,000, respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or UlO RNA. Nuclei of metazoan cells contain a set of small RNAs, the U-type small nuclear RNAs (snRNAs), which are complexed with several distinct proteins to form small nuclear ribonucleoprotein particles (snRNPs) (see refs. 1 and 2 for recent reviews). The snRNAs U1-U6 are extremely abundant in metazoans (about 105_106 copies per cell) and, with the exception of U6 RNA, contain a characteristic 2,2,7-trimethylguanosine (M3G) cap structure at their 5' termini. U6 RNA is packaged into one snRNP with U4 RNA, whereas U1, U2, and U5 RNAs are organized into monomeric particles (3-5). These snRNPs, except for U3, are immunoprecipitated by antibodies from patients suffering from connective tissue diseases. Anti-Sm antibodies recognize common proteins present in all particles identified so far, whereas proteins characteristic for U1 or U2 snRNP are recognized either by patient sera or by monoclonal antibodies raised against purified U1 snRNP (reviewed in ref. 1). In addition, the particles can be precipitated by antibodies directed against the m3G cap structure (6-8).