Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome

The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.”     

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.     

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.     

An improved rapid enzymatic method of RNA sequencing using chemical modification.

A version of rapid gel sequencing procedure based on the analysis of partial endonuclease hydrolizates of chemically modified 5'-32P-labelled RNA is suggested. Complete and selective modification of cytidilic residues by a methoxyamine-bisulfite mixture leads to the unfolding of the RNA secondary structure and, due to this effect, to the generation of a more uniform set of fragments after partial RNAase hydrolysis. The position of cytidines in an RNA sequence can be determined by restricting the hydrolysis of phosphodiester bonds between the modified CMP residues and their 3'-neighbours with T2 and A RNAases. The method was verified with tRNATrp (yeast) and 5S RNA (rat liver and yeast).     

MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS : II. A Comparison of the High Molecular Weight RNA from Mouse and Hamster Cells

Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 10⁶ daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

Nuclear ribonucleases and post-transcriptional changes of RNA. Specificity and other properties of rat liver nuclear endonuclease]

Some physico-chemical properties, specificity and the character of action of rat liver nuclear ribonuclease are studied. The enzyme maximal activity was observed at pH 7.5--8.0, ionic strength 0.02--0.3, Mg2+ being necessary. Nuclease is an oligomer, having molecular weight is 160000--180000 daltons and containing separate associates. Purified enzyme is free of contaminating activities (polynucleotidephosphorylase, DNAse; 5'-nucleotidase, and alkaline phosphatases). It is shown to hydrolyse polyA and RNA for endonuclease type, degradation products being oligonucleotides terminating with 5'-phosphate and 3'-hydroxyl groups. RNAse hydrolyses all phosphodiester bonds in polynucleotides, developing no specificity to the nature of bases. Relative hydrolysis rate for different substrates decreased as follows: polyA greater than yeast RNA greater than polyC greater than polyU greater than 28S rRNA greater than greater than 18S rRNA greater than polyA-polyU. The enzyme may be classified as ribonucleate-5'-nucleotidehydrolase (EC 3.1.4.9.).     

Secondary structure of the 5'-terminal region of the 18S ribosomal RNA5.3S fragment from the rat liver

Two small RNA fragments, 5,3S and 4,7S, were observed in gel electrophoretic analysis of RNA of the 40S ribosomal subunit of rat liver. 5,3S RNA (134-136 nucleotides long) proved to be 5'-terminal fragment of 18S ribosomal RNA, whereas 4,7 RNA is the degradation product of 5,3S RNA with 27-28 5'-terminal nucleotides lost. The secondary structure of 5,3S RNA was probed with two structure-specific nucleases, S1 nuclease and the double-strand specific cobra venom endoribonuclease. The nuclease digestion data agree well with the computer generated secondary structure model for 5,3S RNA. This model predicts that the 5'-terminal part of rat liver ribosomal 18S RNA forms an independent structural domain. The affinity chromatography experiments with the immobilized 5,3S fragment show that 5,3S RNA does not bind rat liver ribosomal proteins.     

The Preparation of Biologically Active Messenger RNA from Human Postmortem Brain Tissue

Abstract: Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)-cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell-free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one- and two-dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two-dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

石斛总RNA提取方法的研究

采用TRIZOL法、异硫氰酸胍法、Tris-硼酸法和改良的RNA提取方法提取石斛的总RNA,并通过凝胶电泳、紫外分光光度法检测提取的RNA样品的品质。研究结果表明:改良的RNA提取方法提取的RNA具有28S rRNA和18S rRNA两条清晰的条带,且无降解。OD260nm/OD280nm接近2.0,具有较高的纯度。其它三种方法获得的RNA品质较差,有降解和弥散现象。将改良的RNA提取方法提取的RNA逆转录成cDNA,经RAPD扩增,出现清晰的条带,进一步证明改良的RNA提取方法提取的RNA具有很高的纯度,可以满足进一步分子生物学研究的要求。