Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Size and location of poly (A) in encephalomyocarditis virus RNA.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.     

More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA.

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.     

The properties and function of rapidly-labelled nuclear RNA

Summary Nuclei were isolated from cultured cells of Acer pseudoplatanus L. previously pulse-labelled with [5-³H]uridine or [³²P]phosphate and the properties of the rapidly-labelled RNA were studied. Polyacrylamide gel electrophoresis showed ribosomal RNA precursors and processing intermediates with molecular weights of 3.4, 2.5, 1.4 and 1×10⁶ daltons, together with polydisperse RNA. The relative proportions of ribosomal RNA precursors and polydisperse RNA varied according to the length of the labelling period, but after 30 min approximately 90% of the radioactive RNA was polydisperse. The relationship between this polydisperse RNA and messenger RNA was investigated. The percentage of total nuclear RNA retained by chromatography on oligodeoxythymidylic acid-cellulose columns varied from 6% to 16% depending on the length of the labelling period. This RNA fraction, which has an adenylic acid content of approximately 45%, is assumed to represent RNA with polyadenylic acid sequences attached. A larger proportion of the nuclear polydisperse RNA lacked polyadenylic acid. Both types of polydisperse RNA were similar in size and during polyacrylamide gel electrophoresis migrated as broad peaks with an average molecular weight of approximately 10⁶ daltons. The polydisperse nuclear RNA that lacks polyadenylic acid was found to be similar in nucleotide composition to ribosomal RNA and is assumed to represent growing chains of ribosomal precursor RNA. After short labelling times the majority of the radioactivity incorporated into nuclear RNA is present in molecules of this type. This suggests that the designation of pulse-labelled polydisperse RNA as messenger RNA or precursor to messenger RNA solely on the basis of rapid labelling and size heterogeneity is unsound. The average molecular weight of the polyadenylic acid-containing messenger RNA from the cytoplasm was less than that of the corresponding nuclear RNA (6 and 9×10⁵ daltons respectively). This suggest either that the majority of the nuclear polyadenylic acid-containing RNA does not enter the cytoplasm, or if it does, that it first undergoes a reduction in size.Abbreviations rRNA ribosomal RNA - mRNA messenger - RNA poly(A), polyadenylic acid, poly(A) and poly(A) - RNA RNA with and without poly(A) sequences attached - poly(U) polyuridylic acid - oligo (dT)-cellulose cellulose with oligo deoxythymidylic acid covalently attached - C cytidylic acid - A adenylic acid - G guanylic acid - U uridylic acid     

In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.     

Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA

Poly(A)-containing RNA isolated from the components of a HeLa cell mitochondrial lysate which sediment in the polysome region of a sucrose gradient have been analyzed for the presence of discrete species. Eight distinct components have been identified by polyacrylamide gel electrophoresis after formaldehyde treatment. These components, which are highly reproducible in their occurrence and relative amounts under widely varying conditions of isolation, have been characterized as to their sedimentation behavior under denaturing conditions, poly(A) content and homology to separated strands of mitochondrial DNA.One of the discrete components was previously shown to have a sedimentation coefficient of about 7 S in the native state and a molecular weight of about 9.0 × 10⁴, as estimated from its sedimentation rate in formaldehyde. The molecular weights of the other seven components, as derived from sedimentation data, range between 2.6 and 5.3 × 10⁵.The 7 S RNA is complementary to the light mitochondrial DNA strand, while the other seven components are complementary to the heavy strand. Together with the two mitochondrial rRNA species and with mitochondrial 4 S RNA, the eight poly(A)-containing RNA components, if distinct in sequence, would account for about 70% of the single-strand informational content of HeLa mitochondrial DNA.     

Membrane-bound mitochondrial DNA: isolation, transcription and protein composition.

Mitochondrial membrane-bound DNA complex from bovine heart mitochondria lysed in the presence of Triton X-100 was isolated by differential centrifugation. The yield of "nucleoid" is about 30 microgram protein/mg mitochondrial protein. It contains about 3-5 microgram DNA/mg protein and varying amounts of RNA. The heart mitochondrial nucleoid actively synthesizes RNA. The nucleoid fraction contains about sixteen different proteins as evidenced by urea-SDS gel electrophoresis and about twenty-one proteins as evidenced by acid-urea gel electrophoresis. It appears that the nucleoid is attached to the inner membrane since it does contain cytochromes.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)-cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)-cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one-half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)-containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA-precipitable material in a cell-free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

Discrete poly(A)-RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5′-termini

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

The segments of influenza viral mRNA.

Influenza viral mRNA, i.e., complementary RNA (cRNA), isolated from infected cells , was resolved into six different species by electrophoresis in 2.1% acrylamide gels containing 6 M urea. The cRNA''s were grouped into three size classes: L (large), M (medium-size), and S (small). Similarly, when gels were sliced for analysis, the virion RNA (vRNA) also distributed into six peaks because the three largest vRNA segments were closely spaced and were resolved only when the gels were autoradiographed or stained. Because of their attached polyadenylic acid [poly(A)]sequences, the cRNA segments migrated more slowly than did the corresponding vRNA segments during gel electrophoresis. After removal of the poly(A) by RNase H, the cRNA and vRNA segments comigrated, indicating that they were approximately the same size. One of the cRNA segments, S2, was shown by annealing to contain the genetic information in the vRNA segment with which it comigrated, strongly suggesting that each cRNA segment was transcribed from the vRNA segment of the same size. In contrast to the vRNA segments, which when isolated from virions were present in approximately 1:1 molar ratios, the segments of the isolated cRNA were present in unequal amounts, with the segments M2 and S2 predominating, suggesting that different amounts of the cRNA segments were synthesized in the infected cell. The predominant cRNA segments, M2 and S2, and also the S1 segment, were active as mRNA''s in wheat germ extracts. The M2 cRNA was the mRNA for the nucleocapsid protein; S1 for the membrane protein; and S2 for the nonstructural protein NS1.     

Overestimates of the size of poly(A) segments.

Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.     

cDNA cloning,functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.