Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein

A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing. In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing. Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease. The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis. The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length. Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP. RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981). Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III. Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein. These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP. The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.     

Messenger ribonucleoprotein particles in silkmoth oogenesis

Silkmoth oocytes contain significant amounts of nontranslating cytoplasmic messenger RNA apparently stored until fertilization. The physical state of this mRNA was examined by bouyant density centrifugation on cesium chloride gradients. Messenger elements were isolated either by oligo(dT) cellulose chromatography or by separation of ooplasm on sucrose gradients. After CsCl density gradient centifugation the mRNA particles banded in a region (1.42–1.48 g/c³) which would indicate a substantial protein content. Electron microscope examination of mRNP fractions revealed particles ranging in size from 180–250 Å.     

Response of cell surface glycosyl transferases to dibutyryl adenosine-3',5' cyclic monophosphate in virus-transformed and normal cells.

Galactosyl and sialyl transferases in the plasma membrane of SV40-transformed mouse cells were inhibited by 0.5 mM dibutyryl adenosine-3′,5′ cyclic monophosphate (db-cAMP) while those of normal cells did not respond to this compound. The differential effects of dibutyryl adenosine-3′,5′ cyclic monophosphate on the membrane-bound glycosyl transferases were observed both in isolated plasma membrane and in intact cell membrane. It is suggested that some of the morphological restorations of normal cell characteristics during reverse transformation are partly due to the direct effect of this compound on the cell membrane.     

Clustering of the DNA sequences complementary to repetitive nuclear RNA of HeLa cells.

We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.     

Levels and modification of methionyl-transfer RNA in preimplantation rabbit embryos.

Transfer RNA with l-methionine acceptor activity was extracted from preimplantation rabbit embryos and purified on reverse-phase-3 columns. The molar quantity of methionine acylated to RNA increases as embryo development proceeds from the 16-cell stage to the 80,000 cell blastocyst stage. However, the quantity of methionyl-tRNA per genome declines 100-fold as the embryo cell number increases. Formylation of methionyl-tRNA illustrated that approximately one-third of tRNA^Met extracted was tRNA_f^Met. Methylation of purified methionyl-tRNA by an adult rabbit liver methylase extract illustrated that two-day preimplantation embryo tRNA is highly hypomethylated relative to tRNA from later stages of development. The hypomethylated methionyl-tRNA was also less effective in ribosome binding studies than more fully methylated methionyl-tRNA present in the later stages of embryo development.     

Subunits of RNA polymerase in function and structure; Maturation in vitro of core enzyme from Escherichia coli

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α₂β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α₂ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α₂ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.In the course of activation of the premature core complex, the subunit σ interacts with both the α₂β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.     

Stimulation by polydeoxyribonucleotide of histone phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase.

[³H]Dihydroalprenolol bound to a single population of high affinity sites in rat myocardial membranes when the concentration of the radioligand was below 5 nM. These sites displayed characteristics which would be expected of binding to the β-receptor. Kinetic- and Scatchard-derived dissociation constants were 0.6 and 2.0 nM, respectively. Binding was to a limited number of sites, 60 fmols/mg protein. Scatchard analysis using radioligand concentrations in excess of 5 nM resulted in concave upward plots suggestive of more than one population of binding sites. The lower affinity sites (labeled at high radioligand concentration) were non-stereospecific in nature and became a progressively larger fraction of “specific binding” as the concentration of dihydroalprenolol was increased above 5 nM.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

Identification of the small subunit of ribulose 1,5-bisphosphate carboxylase as a product of wheat leaf cytoplasmic ribosomes.

Polyribosomes from greening wheat seedlings (Triticum vulgaris) were allowed to incorporate [³H]leucine into proteins in the presence of a wheat germ supernatant fraction under conditions permitting the completion and release of polypeptides by cytoplasmic polyribosomes. The released proteins were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Discrete proteins, as well as a variety of poorly resolved proteins, were observed to have been labeled. The molecular weight distribution of the labeled proteins correlated well with the distribution of polyribosome size classes present in the samples. Neither the large nor the small subunit of ribulosebisphosphate carboxylase were detected as labeled peaks by this procedure.Immune precipitates formed by the addition of carrier small subunit, detergent, and anti-small subunit serum to the released proteins contained a substantial proportion of nonspecifically precipitated material resembling the population of released proteins, but they also contained two discrete peaks not resolved previously, one having mobility slightly faster than the light chain of immunoglobulin (20,000 daltons) and the other having mobility identical to that of small subunit carrier (ca. 12,000 daltons). Samples containing the latter material were shown to contain labeled tryptic peptides corresponding to those of the small subunit carrier. The results establish that the small subunit of ribulose bisphosphate carboxylase is the product of a small proportion of cytoplasmic polyribosomes during greening of etiolated wheat seedlings.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.     

Cell-free Cytoplasmic Polyadenylation of Oogenic RNA

The products of cell-free ATP incorporation mediated by cytoplasmic fractions prepared from unfertilized sea urchin eggs, anucleate egg halves, nucleate egg halves, emetine-treated fertilized eggs, and four-cell embryos have been characterized to determine to what extent the polymers synthesized are poly(A) and to assess the size distribution of the primers adenylated. As judged by alkaline lability, ribonuclease resistance, and retention on poly(U)-impregnated filters, greater than 92% of the label recovered after RNA extraction is present in poly(A). LiCl fractionation indicates that little, if any, free poly(A) is synthesized or cleaved from RNA primers during the reaction, and that 4S RNA is not an effective initiator. In excess of 85% of the poly(A) is associated with RNA having S-values greater than or equal to 18S. Sedimentation profiles of RNA adenylated in the unfertilized egg and anucleate egg half reactions are identical. Suppression of in vivo protein synthesis by emetine alters the profile of RNA subsequently adenylated in vitro. It is proposed that the apparent constraints on the utilization of cytoplasmic RNA or ribonucleoprotein primers of oogenic origin may be effected by RNA-associated proteins capable of regulating the selection and/or extent of their polyadenylation during early embryogenesis.     

The regional metabolism of 5-hydroxytryptamine in mouse brain in vitro.

The relative rates of formation of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) from exogenous 5-hydroxytryptamine, showed regional variations when examined in homogenates of seven separate areas of mouse brain. 5-HTOL production was highest in the cerebellum, and lowest in the corpus striatum, whereas the production of 5-HIAA was greatest in the hypothalamus. Addition of NADPH was shown to increase the formation of the alcohol catabolite in whole brain homogenates. The production of 5-HTOL decreased in the brain homogenates of mice which had previously been injected with phenytoin sodium or oxypertine, with the latter also causing a fall in overall 5-HT metabolism.     

Sea urchin embryo chromatin and nuclear ribonucleoproteins fractionated by anion exchange chromatography.

Chromatin and ribonucleoproteins released from sea urchin embryo nuclei were characterized on the basis of sedimentation properties, buoyant densities and fractionation by anion exchange chromatography. DEAE- and ECTEOLA-cellulose chromatography was used to assay nuclear purity, insofar as ribosomes and polyribosomes could be readily distinguished from ribonucleoproteins released from nuclei. This chromatography was used to separate chromatin fragments on the basis of DNA size, to prepare chromatin fragments substantially enriched in nonhistone proteins, and to analyze nuclear ribonucleoproteins. Solubilized chromatin is fractionated into major and minor components by ECTEOLA-cellulose chromatography. The DNA of these chromatin fractions was analyzed with respect to buoyant density and hybridization with nuclear RNA.