CORRESPONDENCE

THE MEASUREMENT OF MESSENGER RNA STABILITY: HAS ANYONE EVER SEEN “ACTIVATOR” RNA?

Originaf article: The regulation of gene expression in eukaryotic cells. Gerald M. Kofodny. Medical Hypotheses 1, 1.5, 19 75.

Comment by TOM SENSKY, Department of Biochemical Pathology, University College Hospital Medical School, London WC 1E 655, England.

Messenger ribonucleic acid (mRNA) stability has been largely ignored in models of gene expression. Kolodny’s recent suggestions (1) are to be welcomed in this respect as exceptional, in the midst of the widespread reluctance even to interpret fully data on mRNA stability. Such reluctance is however understandable; when HeLa MRNA can have an apparent half-life of anywhere between three hours (2) and three days (3), it is small wonder that would-be hypothesizers despair (cf. the ‘Hypotheson’ (4)). What is perhaps more surprising is the extraordinary reluctance there appears to have been even to discriminate between these widely differing estimates of stability, with some exceptions (e.g. 5).

One cont~buting factor * to x this ~fo~unate situa~on has undoubtedly been the varied, and often inadequate, methodology used to attempt to assess mRNA stability. At the root of this, lie the nucleotide pools, about which most biochemists seem never to have heard. Those who have heard never seem to agree, and in any case seem to live in the hope that if ignored, the pools will very rapidly go away. This has formed the basis of one approach to the measurement of mRNA half-life--most common methods arguably represent no more than different approaches designed to cope with the behaviour of the RNA precursor pools by different means. In this context, however, ignorance seems to have become infectious. The use of Actinomycin D (6), for example, certainly should control incorporation into RNA of radioactive precursors, allowing effects of the pools to be ignored (7). At the same time, other effects of this antimetabolite (8) cannot be overlooked, as has often been the case. One of these effects is the interference with the transfer of RNA from cytoplasm to nucleus (9) (cf I). Other attempts to ignore the precursor pools, such as those which involve subjecting cells to treatment at low temperatures (3, IO), also have potentially unfavourable consequences ( 11, 12), whose possible effects on RNA synthesis and stability cannot be overlooked simply because too little is known of them.

One of the reports cited by Kolodny in support of polysomal ‘activator’ RNA (1) is thus suspect on these grounds, since Actinomycin D was used (7). This is

perhaps just as well, since if mRNA fz$erime (7) were translated into mRNA gulfs (13), about 80% of polysomal mRNA would be almost as unstable as HnRNA. Apart from suggesting the mRNA has two components with different half-lives, the other report cited by Kolodny (14) has very little in common with this first-the half-lives are longer, and the estimated proportions of the two components are quite different. However, here there is the suggestion that the more stable component makes up two-thirds of the steady-state concentration of polyadenylated polysomal RNA (14). If all this were ‘activator’ RNA, such a component would presumably make up a major fraction of each individu~ mRNA molecule. To remain consistent with the concept of ‘activator’ RNA (1) under such circumst~ces, one would have to explain how furge tracts of repetitive RNA sequences in mRNA could have been overlooked (15), or alternatively to postulate that the sequences at the 5’ end of HnRNA contained equally large and therefore conspicuous non-repetitive RNA tracts, which have not yet been found

(16). It must also be remembered that most of the reports on

mRNA stability that do not involve ‘specialized’ and arguably atypical messengers (17) have been derived from total ~ly~enylat~ polysomal RNA from an asynchro- nou~y-~ow~g cell ~p~ation (13, 18). Such methods are ~doubt~y undoubted for the detection of patterns .of mRNA stability (13, 14) and the assessment of the ‘magnitude of the problem’, but they too have limitations that must not be overlooked. The assumptions used to derive results from data from such sources (13, 19) might hold only because they are applied to what is in effect the statistical analysis of a large number of perhaps widely different individual messengers, taken from a highly selected population. Polyadenylated polysomal RNA, for example, is not the same as total polysomal RNA (18), which might in turn be heterogen~us in terms of its stab~ty and thus different from total c~opl~mic mRNA (20). There is no a priori reason why synthesis of individual messengers should follow zero-order kinetics, even though the synthesis of total polyadenylated

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polysomal RNA appears to do so (13, 18). In fact, commoniy accepted ideas about eukaryotic transcriptional control (21) imply that mRNA is transcribed from DNA in a non-random, possibly sequential manner, as is the case in prokaryotes (22) and has been suggested for some tower eukaryotes (231, If this is the case, even the concept of a ‘steady-state’ concentration would be meaningless when applied to individual messengers, particulz+rly if the apparent (statistical) half-life of the bulk of polysomal mRNA in growing cells was in the range of one generation time (5, 13, 18). The concentration of individual messengers could under such circumstances vary

considerably within one cell cycle, and might even become rate-limiting in protein synthesis (cf 14). mRNA av~ab~ty has been shown to be the rate-limiting factor in certain stages of ovalbumin synthesis in chick oviducts (24). This may be a ‘specialized’ system, but it does represent one of the few that has been thoroughly investigated.

The refinement of new techniques currently under development (eg 25) might bring the study of individual messengers in general within access, It is likely that only in this way will a complete account be possible of the role of mRNA in the process of gene expression. In the meantime, Kolodny may well be righ% the results on mRNA stability (6, 14) which he quotes form only a small part of the evidence presented in favour of his hypothesis (1). Further- more, nothing has been said above to refute this hypothesis. If ‘activator’ RNA does indeed for part of polysomal mRNA, it is has never yet been seen or described as a distinct species.

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J& :;xh& &La cells. P&c Nat Acad Sci 7& IIS, 1973.

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Barr HJ, A new genetical Biol 3, 5 14, 1962.

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Wiegers U, Kramer 0, Kla proth K, Rehpenning W, Hilz H, Determination of mRNA h a& -bfe in HeLa cultures by a poly(A)_ independent direct analysis of speci8c radioactivity of mRNA., Eur J B&hem SO, 557, 1975. Cheevers WP, Sheinin R, Selective rn~~~ent of the s nthesis and metabolic stabilitv of rness~a~ RNA in 3T3 ceils. t;* Biophys Aeta 204, 4?9, 1910. -

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Perry RP, Selective diects of Actinomycin D on the intracellular distribution of RNA synthesis in tissue culture cells. Exp Cell Res 29, 400, 1963. Soiero R, Amos H, Messenger RNA half-fife measured by use of A~orny~~ D in animal &&-a caution. Biochim Eophys Acta 129, 406, 1966. Goldstein L, Wise GE, Beeson M, Proof that certain RNA’s shuttie ~~-r~9~~y betwmn cytoplasm and nucleus. Exp Cell Res 76,

Schbitisse~ C, Detection of an unstable RNA in chick fibroblasts a&r reduction of the UTP pool by glucosamine. Bur J Biochem zr, 358, 1971.

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Lindahl PE, Siirenby L, A new method for the continuous selection of &Is in mitosis. Exp Cell Res 43, 424, 1966. HoleEkovii E, Baudyzovii M, Cinnerovri 0, Adaptation of mamrn~i~ cells to the cold. Exe CeU Res 40. 396. 1965. Greenberg JR, High stability df messenger kNA’ in growing cultured cells. Nature 240. 102. 1972. Singer RH, Penman S, Messenger RNA in HeLa celfs: kinetics of formation and decay. J Mel Biol 78, 321, 1973, Dina D, Mezza I, Crippa M, Relative positions of the ‘repetitive’, ‘uni Mel oy ‘i

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(A) fragments of mRNA. Nature 248,486, 1974. Je mek W, Salditt M, DarneIl JE, Arrsngement of r

specific otigonucleotid~ within poly(A) terminated HnRNA molecules. Ceff 1, 43, 1974. Kafatos FC, mRNA stability and cbll&+r d~erentiation, F‘ tk K~r~~ins~ Sym~si~rn~ Trans~~~~ion in ~e~r~~c~~ve Tissue, P ed 1 E. Diczfalusy Karolinska Institutet, Stockhdm, 1972.

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Perry RP, Kelley DE, Messenger RNA turnover in mouse L cells. J Mel 79, 681, 1973, Spradling A, Hui HZ penman S Two very different components of messenger RNA 1x1 an insect cell line. Cell 4, 131, 1975. Cowan NJ, Mifstein C, Stability of ~ytop~~rni~ ribonu&i~ acid in a mouse myeloma: estimation of the half-life of the messenger RNA coding for an immunoglobulin Iight chain. J Mol Biol 85 469. 1974. BriGen RJ, Davidson EH, Gene regulation for higher ceils: a theory Science 165, 349, 1969. Jacob F, Monod J, Genetic regulatory mechanisms in the synthesis of proteins. J Mel Biol 3, 318, 1961. Tauro P, Halvorson HO, Epstein RL: Time of gene expression in relation to centromere distance durm8 the ceil cycle of Sac- ckaromyces cerevisciae. Proc Nat Acad Sci 59, 277, 1968. Palmiter RD, Quantitation of parameters that determine tbe rate of ovalbumin synthesis. Cell 4, 189, 1975. Schechter I. Use of antibodies for the isolation of bioloaicallv oure messenger hbonucleic acid from fully functional euk&otic’ &is. Biochem 13, 1875, 1974.

Rep& from Gerald M. Irl0lodB.y I would in general agree with Dr, Sensky’s caution

regarding mRNA stability in the light of consideration of nucleotide pools, actinomycin D effects and Low t~~rat~~. However, one important ~ncIusion from the studies thus far reported is that mRNA does appear to have components which differ in metabolic stability. We have recently reported component of ribosomal RNA which differ in metabolic stability (1). The more stable components could represent the primer RNA required by my hypothesis, Dr. Sensky is of course correct that activator RNA has not yet been demonstrated. This will require degradation of a purified metabolically active RNA followed by an unequivocal demonstration that a degradation product has been incorporated into a newly synthesized rneta~~~~y active RNA. We are currently attempting such experiments using ribosomd RNA rather than messenger RNA simply because of the relative ease of isolating rRNA in comparison with mRNA.

1. Kolodny G M. Turnover of Ribosomal RNA in Mouse Fibroblasts (3T3) in Culture. Exp Cell Res. 91, 101, 1975.

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