Proteins crosslinked to messenger RNA by irradiating polyribosomes with ultraviolet light.

A set of proteins crosslinked to L-cell mRNA by irradiating polyribosomes with 254 nm ultraviolet light has been identified. 35S-methionine-labeled crosslinked mRNA-protein complexes were isolated by chromatography on oligo(dT)-cellulose under conditions that prevented non-covalent binding of proteins to RNA and to the column. After enzymatic removal of the RNA the proteins were analyzed in sodium dodecylsulfate/polyacrylamide gels. Six proteins having molecular weights of 98,000, 78,000, 75,000, 68,000, 62,000, and 52,000 were crosslinked to mRNA whether intact polyribosomes or EDTA- released mRNA-protein complexes were irradiated. Digestions with specific RNAases and chromatography on oligo(dT)-cellulose were used to show that a protein of 78,000 daltons was the only one crosslinked to poly(A), and the other proteins were crosslinked to sequences other than poly(A). However, a 78,000 dalton protein was also crosslinked to a sequence other than poly(A).     

Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography.

A study has been made of the developmental changes that occur in the RNA and protein moieties of mRNA-protein particles isolated from newborn and adult rat forebrain free polyribosomes. mRNA-protein particles were isolated by oligo(dT)-cellulose chromatography from salt-washed polyribosomes dissociated by puromycin/0.5 M-KCl treatment as two fractions (E1 and E2) by using Tris/HCl/NaCl eluting buffers containing respectively 25 and 50% (v/v) formamide. Isopycnic centrifugation on CsCl gradients showed that the newborn-derived fractions E1 and E2 has buoyant densities of 1.48--1.50 and 1.41--1.43 g/cm3. Adult-derived E1 and E2 fractions had corresponding values of 1.47 and 1.42 g/cm3. The pooled mRNA-protein particles from the E1 and E2 fractions after deproteinization with proteinase K sedimented with a mean size of approx. 18 S on a sucrose gradient containing 85% formamide with little differences between mRNA molecules from newborn and adult. The mean lengths of the poly(A) segments were similar, being about 130 nucleotides long. Distinct changes were found in the protein composition of the mRNA-protein particles. Fractions E1 and E2 from the newborn contained two major proteins of mol.wts. 74 000 and 52 000 with differences in the relative proportions in each fraction. In contrast, adult fractions E1 and E2 contained predominantly the larger protein. However, the adult fraction E2 contained a more heterogeneous population of minor bands of proteins, including that of mol.wt. 52 000. The findings are discussed briefly in relation to other changes in the developing brain.     

In vivo cross-linking of proteins to mRNA in human cells

Human KB cells were irradiated with ultraviolet light to cross-link mRNA to its associated proteins. More than 75% of both the poly(A)-containing and the poly(A)-lacking mRNAs were cross-linked to proteins after 3 min irradiation. Glycerol gradient analysis showed that no significant RNA chain breakage occurred during this treatment. Cross-linked poly(A)-containing mRNA-protein complexes were purified by oligo(dT)cellulose chromatography in the presence of sodium dodecylsulphate. CsCl gradient analysis revealed that the low salt eluted particles had a buoyant density of about 1.47 g/cm³. To determine which proteins were cross-linked to mRNA, covalent mRNA-protein complexes, labeled in their RNA moiety, were exhaustively treated with nucleases. Polyacrylamide gel analysis showed that most of the residual RNA-radioactivity was covalently bound to proteins of 73000, 69000 and 52000 molecular weight.     

Resonance Raman spectra of heme a derivatives. Evidence for the reaction of peripheral formyl group with HCN and NaHSO3.

A separate and distinct population of polyribosomes exists in the detergent-washed nuclei of adenovirus-infected HeLa cells. These polyribosomes, released by exposure to polynucleotides such as high molecular weight nuclear RNA or poly(U), do not appear to be cytoplasmic contaminants. Nuclear polyribosomes have a considerably lower buoyant density compared to cytoplasmic ones. Nuclear polyribosomes, in a cell-free system of protein synthesis, are six- to eight-fold less active compared to cytoplasmic ones and are insensitive to aurin tricarboxylic acid. They do not complement cytoplasmic polyribosomes in protein synthesis in the cell-free system. Finally, the number of proteins synthesized by nuclear polyribosomes is higher compared with that synthesized by the cytoplasmic ones. Only the virus-specific proteins, including P-VII, are synthesized by cytoplasmic polyribosomes. Nuclear polyribosomes, on the other hand, synthesize virusspecific proteins, including P-VII and VII, and a number of additional proteins not synthesized by the cytoplasmic ones.     

Different complexes are formed on the 3'' end of histone mRNA with nuclear and polyribosomal proteins.

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts.     

In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA-binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal-mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78-kDa protein could be cross-linked to poly(A).     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.     

mRNA display: ligand discovery,interaction analysis and beyond

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.     

Chicken reticulocyte polysomal messenger RNA-protein complex: absence of bound proteins in most of the coding region of beta globin mRNA.

The 15s globin mRNA-protein complex (mRNP) was isolated from chicken reticulocyte polyribosomes dissociated in EDTA. To determine protein binding sites, the mRNP was treated with micrococcal nuclease and the nuclease resistant RNA was mapped to the beta globin gene at the nucleotide level. As far as we can determine there is no bound protein from the Cap site to the poly A addition site of beta globin mRNA in the mRNP except for a short area in the coding region near the translation initiation site.     

Selection of proteins with desired properties from natural proteome libraries using mRNA display

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

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.     

Polysome-associated proteins in herpes simplex virus-infected cells

In the cytoplasm of eucaryotic cells, mRNA is associated with proteins. These mRNA-protein complexes, termed messenger ribonucleoprotein (mRNP) particles, are divided into two functional classes. The first class contains free (non-ribosome-associated) mRNPs which have been termed informosomes by others. The second class of mRNPs, those associated with polysomes, are actively engaged in protein synthesis and are termed polysomal mRNPs. The experiments described in this paper examined the proteins associated with polyribosomes in uninfected and herpes simplex virus type 1-infected cells. The data indicate that after infection with herpes simplex virus type 1, specific changes occur in the proteins which normally are found associated with these polysomal mRNPs. These changes include both the appearance of new and possibly virus-specific proteins and the loss of normal host-specific proteins. The relationship of these changes to the patterns of protein synthesis in these cells is also discussed.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.     

Eukaryotic elongation factor Tu is present in mRNA-protein complexes

By two-dimensional gel electrophoresis, partial peptide mapping, and antibody binding we have shown that eukaryotic elongation factor Tu is in close contact with mRNA in rabbit reticulocytes. It can be crosslinked to mRNA by irradiating both polysomes and 40-80 S mRNA-protein complexes with short-wave UV light. To our knowledge this is the first case in which a known translation factor has been shown to be associated with mRNA in native ribonucleoproteins.     

The mobilization of maternal histone messenger RNA after fertilization of the sea urchin egg

The extent of protein, RNA and DNA synthesis in early cleavage stages of the sea urchin embryo (Parechinus angulosus) was determined. A histone mRNA specific cDNA was used in hybridization experiments to investigate the cytoplasmic localization of maternal histone mRNA in the unfertilized sea urchin egg and first cleavage stage embryo. In the unfertilized egg histone mRNA was localized exclusively in ribonucleoprotein particles with none in ribosomes or polyribosomes. This distribution changed after fertilization, in particular, coupled with the first cleavage telophase there was a significant transfer of histone mRNA from the ribonucleoprotein fraction to the polyribosomes. The results indicate mRNA specific translational control mechanisms.