Polysome Preparations Research Articles

A post-transcriptional regulatory landscape of aging in the female mouse hippocampus.

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3'-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases.

Polysome Preparation, RNA Isolation and Analysis.

During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

Characterization of HULC, a Novel Gene With Striking Up-Regulation in Hepatocellular Carcinoma, as Noncoding RNA

Recent studies have highlighted the role of noncoding RNAs (ncRNAs) in carcinogenesis, and suggested that this class of genes might be used as biomarkers in cancer. We searched the human genome for novel genes including ncRNAs related to hepatocellular carcinoma (HCC). An HCC-specific gene library was generated and screened for deregulated genes with 46 HCCs, 4 focal nodular hyperplasias, and 7 cirrhoses utilizing cDNA arrays. Sequencing of library clones identified a novel ncRNA as the most up-regulated gene in HCC. This gene was also cloned from different monkeys and characterized by quantitative RT-PCR, Northern blot analysis and in situ hybridization. Structural and functional studies included comparative sequence and protein expression analyses, quantitative RT-PCR of polysomal preparations, and siRNA-mediated knockdown experiments. The most up-regulated gene in HCC named highly up-regulated in liver cancer (HULC) was characterized as a novel mRNA-like ncRNA. HULC RNA is spliced and polyadenlyated, and resembles the mammalian LTR transposon 1A. It does not contain substantial open reading frames, and no native translation product was detected. HULC is present in the cytoplasm, where it copurifies with ribosomes. siRNA-mediated knockdown of HULC RNA in 2 HCC cell lines altered the expression of several genes, 5 of which were known to be affected in HCC, suggesting a role for HULC in post-transcriptional modulation of gene expression. HULC is the first ncRNA with highly specific up-regulation in HCC. Because HULC was detected in blood of HCC patients, a potential use as novel biomarker can be envisaged.

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.

Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase.

We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress. Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations. RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific. Unwinding of partially duplexed RNA proceeds in the 5'-->3' but not the 3'-->5' direction using standard assay conditions. Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide. We propose that CrhC unwinds cold-stabilized secondary structure in the 5'-UTR of RNA during cold stress.

INTERACTION OF PLANT POLYSOMES WITH THE ACTIN CYTOSKELETON

Protein composition and functional activity of various polysome subpopulations isolated from Vicia faba L. leaves and Triticum aestivum L. and Hordeum vulgare L. seedlings were studied. Membrane- and cytoskeleton-bound polysomes were more active in the wheat germ cell-free translational system than free polysomes. Several non-ribosomal proteins were detected in the polysome preparations by gel electrophoresis and Western blot analysis: (1) a canonical actin of mol wt 42kDa; (2) a 40kDa protein, demonstrating affinity for ribosomes, sharing some determinants with actin, and present predominantly in the subpopulations of bound polysomes; and (3) an acidic ribosome-associated p40 evenly distributed between free and bound polysomes. The possibility of involvement of these proteins in interactions between polysomes and the actin cytoskeleton is discussed.

Polysomes fromVicia fabaL. leaves bound to the actin cytoskeleton

The presence of polymeric actin in the polysome preparations from broad bean leaves (Vicia faba L. var. Russkie chernye) was detected on dot blots and under the electron microscope using the phalloidin-colloidal gold marker. The fractions enriched in the cytoskeleton-bound polysomes were isolated using different sensitivity of the membranes and cytoskeletal elements to nonionic and ionic detergents. The distribution of a polypeptide with a molecular weight of about 40 kDa in the subcellular fractions was studied and an enrichment of this polypeptide was observed in membrane-bound and especially cytoskeletal-bound polysomes. This polypeptide gave a positive signal when probed with anti-actin serum on Western blots.

The effect of irradiance on the protein synthetic machinery of Vicia faba L. leaves

An increase in irradiance from 10 to 125 W/m 2 resulted in the activation of RNA polymerase I and recruiting of monosomes into polysomes in the leaves of Vicia faba L. Leaf discs from plants grown under 10 W/m 2 synthesised protein in vivo at a higher rate than discs from plants grown under 125 W/m 2, especially when calculated per dry weight basis. The translation activity of polysomes in the cell-free system was also significantly higher in the preparations from plants grown under 10 W/m 2. A search for possible regulators of polysome translation activity resulted in the detection of two major non-ribosomal polypeptides with M r of about 42 and 55 kDa in the polysome preparations. The abundance of these polypeptides depended on the irradiance and leaf age. Preliminary immunoassays and phalloidin test indicate the presence of polymeric actin in the polysome preparations.

Brain mRNA from infants of aluminium-exposed lactating rabbits

The neurotoxicity of aluminium (Al) involves bundling of neurofilaments, increased chromatin binding and decreased protein synthesis in Al injected rabbits. Thus, using an amphipathic Al ligand, maltol, experiments were carried out to examine whether or not administration of Al to lactating mother rabbits reduces brain protein synthesis in their offspring. Lactating mother rabbits received s.c. injections 3 times weekly of aluminium (Al) maltolate (1 mg Al/kg body wt) or an equivalent weight of maltol, for 4 weeks post-partum. Polysome preparations were obtained from the brain of their infants in order to assess mRNA translation in cell-free protein synthesizing systems. The brain polysomes showed a statistically significant reduction in the incorporation of [ 14C]leucine into protein. The poly (A) + and poly (A) − fractions obtained from these polysomes showed reductions of 44% or more in the incorporation of [ 35S]methionine into protein. A variety of products separated by SDS-polyacrylamide gel electrophoresis all exhibited decreased labelling. These experiments suggest that infant rabbits exposed to a highly neurotoxic form of Al in milk exhibit changes in brain protein synthesis which resemble those in infants injected directly with Al.

CDNA cloning of a hnRNP A1 isoform and its regulation by retinol in monkey tracheobronchial epithelial cells

An isoform of the hnRNP A1 was cloned from a cDNA library of monkey tracheobronchial epithelial (TBE) cells by differential hybridization. The cDNA clone MT77 has an insert of 1756 base pairs and the DNA sequence shares high homology to both human A1 α-type and β-type isoforms with the exception of several differences in the coding and noncoding regions. Like the other two isoforms, MT77 has two polyadenylation sites. A probe prepared from the MT77 clone hybridizes to two message bands at 1.4 and 1.8 kb. Both messages were found in a polysomal preparation, suggesting that both messages are used in A1 protein synthesis. The expression of the A1 gene in monkey TBE cells is stimulated by vitamin A (retinol). The results of nuclear run-on transcriptional assays suggest that this stimulation occurs at the transcriptional level. Furthermore, this effect is not prevented, but superinduced, by cycloheximide. These results suggest that vitamin A may be directly involved in regulating A1 transcription through a mechanism similar to the interactions between the retinoic acid responsive elements and the nuclear receptors of retinoic acid.

Protein synthesis in halophytes: The influence of potassium, sodium and magnesium in vitro

The amino acid (35S-methionine) incorporating activity of an in vitro wheat germ translation system was found to be maximal in 80 to 125 mol m−3 K with 2 to 4 mol m−3 Mg both as the acetate. Substitution of Na for K, or chloride for acetate at concentrations above 80 mol m−3 inhibited incorporation. When the K acetate concentration was raised to 200 mol m−3, no incorporation of radioactive methionine occurred. Translation by polysomes extracted from leaf tissue of S. maritima, supplemented with postribosomal supernatant from wheat germ, showed activity which was optimal in the presence of 225 mol m−3 K acetate and 8 mol m−3 Mg acetate. However, the translation system was not directly comparable with the wheat germ system, as studies with an initiation inhibitor, aurintricarboxylic acid, suggested that the S. maritima system was essentially elongation-dependent, while initiation occurred in the wheat germ system. Elongation-dependent polysomal preparations were extracted from leaves of the glycophytes Pisum sativum, Triticum aestivum, Oryza sativa and Hordeum vulgare, and from the halophytes Atriplex isatidea and Inula crithmoides. Translation by polysomes from the salt-tolerant plants was optimal at higher K and Mg concentrations, than by polysomes from the glycophytes. Furthermore, NaCl was better able partially to substitute for the role of K in polysomal preparations from halophytes than glycophytes.

5'-secondary structure formation, in contrast to a short string of non-preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast.

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5'-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5'-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5'-end of the mRNA (overall delta G = -36.6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.

Isolation of a translation-inhibiting peptide from myocardium

We previously reported that an initial response to acute cardiac stress is temporary translational inhibition. Using an in vitro translational assay to follow activity, we purified a translation-inhibiting peptide (TIP) obtained from control canine hearts and hearts acutely stressed by ascending aortic banding and heat shock for 1 h. We isolated a 17-kDa peptide. The amino acid composition was determined and residues 3-20 were sequenced. Treatment of polysome preparations with TIP shifted the polysome distribution profile to that characteristic of the acutely stressed heart in which monosomes predominate. Translational inhibition acted by suppressing formation of the 80S initiation complex. Suppression curves indicate that translation is inhibited by approximately 60%, with the translation of protein over 30 kDa being highly suppressed. We postulate that translational inhibition is an essential initial reaction to acute stress that allows the cell to redirect energy into vita cell functions that permit long-term adaptation to the stress. We believe that translational inhibition is effected by intracellular activator of TIP.

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.

Efficacy of RNase inhibitors during brain polysome isolation

We have investigated the efficiency of heparin, polyvinyl sulfate and yeast RNA (as competitive RNase inhibitors), liver extract (as crude preparation of liver RNase inhibitors) and DEPC (as irreversible non-competitive inhibitor) for the preparation of rat brain polysomes. Sucrose gradient sedimentation profiles, obtained from PMS, were used to determine the optimal concentration of each inhibitor. Diethylpyrocarbonate, whatever the composition of isolation buffer, was found detrimental for brain polysomes. Most of the other inhibitors where found useless or even harmful. A slight positive effect was observed with heparin 0.75 mg/mL both for total yield and sedimentation pattern. It is concluded that the utilisation of most of the widely used RNase inhibitors is of questionnable effectiveness for brain polysome preparation.

Biosynthesis of type I procollagen. Characterization of the distribution of chain sizes and extent of hydroxylation of polysome-associated pro-alpha-chains.

[3H]Proline-labeled nascent procollagen chains were isolated from chick tendon polysome preparations as peptidyl-tRNA complexes by ion exchange chromatography. Proline hydroxylation of the nascent chains was at least 40% complete, based on radioactive hydroxyproline/proline ratios. These data provide the first direct evidence that hydroxylation of procollagen proline residues does occur on nascent chains. The electrophoretic profiles of [3H]proline-labeled nascent chains and of unlabeled nascent chains visualized by Western blotting with 35S-labeled monoclonal antibodies to the alpha 1(I) N-propeptide or the C-propeptides indicate that there are pauses in the translation of procollagen alpha-chains in the intact cells. Approximately 25% of the radioactivity associated with [3H]proline-labeled polysomes was in fully elongated but underhydroxylated (relative to secreted procollagen) pro-alpha-chains. The association of these completely elongated but only partially modified procollagen chains with the polysome complex may facilitate the carboxyl-terminal interactions which lead to triple helix formation.

Isolation of free and membrane-bound polysomes and mRNA highly active in translation and reverse transcription from small discrete regions of rat brain

A procedure is described for the preparation of total polysomes, membrane-bound and free polysomes and polysomal mRNA from as little as 5 mg or less of brain tissue. These preparations were highly active when tested for translation and reverse transcription in vitro. Using this method, polysomes and mRNA from rat cerebral cortex, cerebellum, hippocampus and hypothalamus were compared. The results showed that membrane-bound polysomes were more active than free polysomes in protein synthesis. The activities of polysomes and mRNA for protein and cDNA synthesis were dependent on the specific brain structures from which they were obtained. Polysomes from cerebellum and hypothalamus incorporated amino acids more actively than those from cerebral cortex or hippocampus, when tested in a reticulocyte lysate system. Cerebellar mRNA also showed the highest activity for cDNA syntehsis as compared to mRNAs from the other three tissues.

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver.

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.

Double-stranded and Single-stranded Subgenomic RNAs from Plant Tissue Infected with Tomato Bushy Stunt Virus

SUMMARY Seven species of dsRNA were isolated from Nicotiana clevelandii plants infected with tomato bushy stunt virus (TBSV). One of these had a length of 4.8 kilobase pairs (kbp), corresponding to the replicative form dsRNA of TBSV genomic ssRNA; the other six were subgenomic RNA with lengths ranging from 3.3 to 0.44 kbp. Analyses of total and polysomal ssRNA from TBSV-infected tissue by gel transfer hybridization with cDNA probes prepared from genomic TBSV RNA revealed genomic RNA and nine shorter species of ssRNA. Six of the latter were shown to be artefacts probably attributable to the interaction between fragments of TBSV RNA and plant RNA. The remaining three had lengths of 1.8, 1.5 and 1.1 kbp and, since they were found in polysome preparations, may be TBSV subgenomic mRNA species; none of these corresponded exactly in length to any of the subgenomic dsRNA.

Comparison of polyadenylic acid and oligouridylic acid messenger RNA associated proteins

Various subclasses of messenger ribonucleoprotein particles were prepared from free cytoplasmic and polysome fractions of rat liver on the basis of the homopolymeric content of the constituent RNA's. Two major proteins were evident in the free cytoplasmic preparations: the poly(A)-binding protein was the major constituent of polyadenylated components and a 60 kilodalton protein was the major protein in oligouridylated components. In addition to the poly(A)-binding protein, the polysome fractions contained a 74 kilodalton protein that was present in all subclasses of particles. With both free cytoplasmic and polysome preparations, chromatography on columns of poly(U)-sepharose separated poly-adenylated mRNP's largely on the basis of the length of the poly(A) tract; mRNP's containing short poly(A) tracts (fragment distribution centered on 34 residues) were not retained by the columns, presumably because of the interaction of the poly(A) with poly(A)-binding protein.