RNA of RNA Tumour Viruses contains Poly A

A heterogeneous RNA fraction with properties resembling those of messenger RNA was identified in mammalian mitochondria. Synthesis of contaminating RNA of nuclear origin was suppressed by treatment with camptothecin. Labeling of the messenger-like RNA is completely inhibited by ethidium bromide, a specific inhibitor of mitochondrial functions.Although mitochondrial protein synthesis resembles that of prokaryotes in several regards, the messenger-like RNA is covalently linked to poly(adenylic acid) [poly(A)J. Poly(A) has thus far been found only in eukaryotic cells. The poly(A) segment has a gel electrophoretic mobility of about 4 S, corresponding to a length of 50-80 nucleotides, and thus resembles in size the poly(A) found in some mammalian viral RNAs. The messenger RNA can be released from the mitochondrial protein-synthesizing structure by treatment with puromycin.Mitochondria of eukaryotic cells have a distinct system of protein synthesis that is quite dissimilar from that found in the cellular cytoplasm. The ribosome-like structure of mitochondria is relatively small (60 S) (1-4). Mitochondrial protein synthesis is sensitive to chloramphenicol, an inhibitor of prokaryotic ribosomal function, and insensitive to cycloheximide, an inhibitor of eukaryotic protein synthesis. NFormyl-methionine has been found in mitochondria and is presumed to initiate polypeptide synthesis as in bacteria (5, 6).These properties of mitochondrial protein synthesis suggested a similarity to prokaryotic rather than eukaryotic mechanisms, and there has been speculation about a possible origin of mitochondria in a primordial prokaryotic symbiont.In this report, we describe an RNA species synthesized in mitochondria with properties that suggest it serves as messenger RNA for mitochondrial protein synthesis. Surprisingly, this RNA contains polyadenylate segments, which have, so far, been found only in the messenger RNA of eukaryotic cells (7)(8)(9)(10)(11) Analysis of Total Mitochondrial RNA. Cellular extracts were prepared and analyzed as described (13). Briefly, cells were washed and then suspended in 1 ml of RSB buffer [10 mM NaCl-1.5 mM MgCl2-10 mM Tris* HCO (pH 7.4)] before they were broken in a Dounce homogenizer. Nuclei were removed by centrifugation at 800 X g for 2 min. The supernatant was again centrifuged at 7700 X g for 8 min. This pellet (mitochondrial fraction) was resuspended in sodium dodecyl sulfate (SDS) buffer and extracted by the hot phenol-SDS method (14). The RNA was analyzed by electrophoresis on 2.7% acrylamide gels for 3.5 hr at 7.5 V/cm. Gels were fractionated as described (15).Analysis of Poly(A)-Containing RNA. Mitochondrial RNA was labeled in the presence of camptothecin, and a crude mitochondrial fraction was prepared. Mitochondria were suspended in MSB buffer [100 mM NaCl-10 mM MgCl2-10 mM Tris*HCl (pH 7.4)] and lysed with 0.5% sodium deoxycholate-0.5% Brij 58. (Brij 58 was obtained from the Hercules Powder, Co.) The lysed mitochondria were layered on a 15-30% MBS-sucrose gradients and centrifuged ...

Section: Resultsmentioning

confidence: 60%

Mitochondrial Protein Synthesis: RNA with the Properties of Eukaryotic Messenger RNA

Perlman

Abelson

Penman

1973

“…Some investigators have suggested that the poly(A) sequences may be involved in the synthesis of viral DNA by reverse transcriptase (8,9,11). Now that it has been determined that the poly(A) sequences are located at the 3'-OH termini of genomic and subunit RNAs, the possibilty that the poly(A) sequences could be the binding sites for reverse transcriptase increases.…”

Section: Discussionmentioning

confidence: 99%

Polyriboadenylate Sequences at the 3′-Termini of Ribonucleic Acid Obtained from Mammalian Leukemia and Sarcoma Viruses

Phillips

Park

Hollis

1974

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T1 digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosinelabeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CC1,COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.

“…Under appropriate conditions, the poly(U) in the resulting hybrid molecules is protected from pancreatic RNase action (unpublished experiments in this laboratory, see also ref. 18). The poly(A) in the complex retains the capacity for hybrid formation with poly(U), and can be assayed in this manner.…”

Section: Detection Of the Poly(a) Complex By Interaction With Labeledmentioning

confidence: 99%

A Particle Associated with the Polyadenylate Segment in Mammalian Messenger RNA

Kwan

Brawerman

1972

128

A structure consisting of poly(A) complexed with other components is released from polysomes by ribonuclease treatment. The poly(A) complex has a sedimentation value of 12-15, while the corresponding sedimentation value for free poly(A) is 4. The complex does not appear to represent an artifact formed by interaction of free poly(A) with either cytoplasmic or polysomal proteins. The polynucleotide released from the complex by treatment with sodium dodecyl sulfate shows the same electrophoretic mobility as that of poly(A) isolated from deproteinized polysomal RNA. The poly(A) in the complex is partially protected from digestion by T2 ribonuclease. At least part of the poly(A) is available for base pairing with poly(U). The components associated with the poly(A) cause it to bind to Millipore filters at low ionic strength. These components are removed from the complex by Pronase digestion. The findings indicate that the poly(A) segment in messenger RNA serves as a binding site for a particle. This particle appears to consist of proteins.