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The poly(A) segment of mRNA: (1) Evolution and function and (2) the evolution of viruses
J. theor. Biol. (1978) 71, 323-338
The Poly(A) Segment of mRNA: (1) Evolution and Function and (2) The Evolution of Viruses RICHARD K. CARLIN University
of Houston, Department of BiophJjsical Sciences, Houston, Texas 77004, U.S.A.
(Received 25 February
1977, and in revisedform
22 August 1977)
The poly(A) segment of mRNA has been shown to be related to evolution. The poly(A) segment increases in length as the complexity of the organism increases. The shortest poly(A) size (1-5 AMP units) exists on mRNA from bacteriophages. The largest poly(A) segments (200-250 AMP units) exist in highly differentiated tissue. The log of poly(A) size and the log of mRNA half-life for different organisms was found to be related in a linear manner. Predictions of specific mRNA half-lives and poly(A) sizes were made to help show a direct relationship. This relationship was also valid for histone poly(A) minus mRNA, provided DNA synthesis is interrupted. Poly(A) segments from viruses reflect the evolutionary status of the viruses. On the basis of viral poly(A) sizes the origin and evolution of viruses is proposed. Important points of this theory are (1) a type of previrus preceded procaryotic cells, (2) these previruses developed into procaryotic cells and DNA bacteriophages, (3) the first eucaryotic viruses contained RNA and originated from procaryotic mRNA, (4) DNA viruses may have developed from RNA viruses as reverse transcriptase developed, and (5) continued evolution resulted in the development of oncogenic viruses.
1. The Poly(A) Segment of mRNA: (1) Evolution and Function and (2) The Evolution of Virnses Poly(A) has been found to be a major component of mRNA (Edmonds & Caramela, 1969; Lee, Mendecki, & Brawerman, 1971). This segment is attached to the 3’ end of mRNA and ranges in size up to 250 AMP units long (Brawerman, 1974). The existence of poly(A) tracts has prompted a great deal of speculation relating to the precise function of these polynucleotide sequences. Possible functions which have been proposed are that poly(A) may be involved in the transport, translation, or stability of mRNA. One method which might aid in the elucidation of poly(A) function was to study its evolution. Table 1 shows the relationship of poly(A) size and the source from which it was obtained. A definite pattern is formed on the basis of the length of 323
0022-.5193/78/0406-0323$.0200/O
0 1978Academic Press Inc. (London) Ltd.
TABLE 1
viridae
Drosophilia & HeLa Achlya ambisexualis, fungus Neurospora crassa, fungus Naegleria gruberi, amebae Cotton
Mitochondria,
Blastocladiella emersonii, fungus Rhizopus stolonifer, fungus Trichoderma viridae, fungus
Chloroplast, maize Chlorella, green algae Yeast Phaseolus areus, mung bean
Caulobacter
Trichoderma
yeast
crescentus
coli
Mitochondria, Mitochondria,
Escherichia
n phage T7 phage 480 phage
Organism
2 50-100 80 75-100
50-60
50 5ot 50 50
:2+
15-50
20-30
25 20-25
2-5
l-5
1-5
Poly(A) size Rosenberg, Weissman & DeCrombrugghe, 1975 Kramer, Rosenberg & Steitz, 1974 Pieczenik, Barrel1 & Gefter, 1972 Nakazato, Venkatesan & Edmonds, 1975 Rosen & Edelman, 1976 Hendler ef al., 1975 Ohta, Sanders & Newton, 1975 Haff & Bogorad, 1976 Scragg & Thurster, 1975 McLaughlin et al., 1973 Higgins, Mercer & Goodwin, 1973 Jaworski, 1976 Freer, Mayama & Etten, 1977 Rosen & Edelman, 1976; Rosen, Edelman & Gallum, 1975 Hirsch & Penman, 1973 Silver & Horgen, 1974 Mirkes & McCalley, 1976 Walsh, Nakazato, Hickey & Edmonds, 1976 Van De Walle, 1973
listed in increasing size
Relationship of poly(A) and evolution. Average newly synthesized poly(A) lengths for dz@erentorganisms have been
pyriformis
gracilis
7 Derived from electrophoretic
Mouse kidney Lymphocyte Barley, aleuron tissue Differentiated neuroblastoma Rabbit brain
Euglena
100 125 120-150 SO-200 150 140-l 60t 15@-160t 160t 14&180 170 130-200 145-210 170-220 200 200 150-200 200-225 225 200-250 200-250 200-250
K120t
90
and sucrose gradient S values.
Pieris brassicae, wing imaginal disk Mouse L cells Chick embryonic cells Drosophila Liver Mosquito HeLa cells Ehrlich ascites Sarcoma 180 Myeloma yiciafaba, meristematic root tissue
Sea urchin
Dictyostelium discoidem, cellular slime mold Physarum polycephalum, unicellular slime mold Xenopus laevis, embryonic cells
Maize Rice, callus tissue
Tetrahymena
Rodriquez-Pousada & Hayes, 1976 Haff & Bogorad, 1976 Manahan, App & Still, 1973 Firtel, Jacobson & Lodish, 1972; Sheiness & Damell, 1973 Adams & Jeffery, 1977 Dina, Meza & Crippa, 1974; Sagata, Shinokawa & Yamana, 1976 Slater, Slater & Gillespie, 1972; Wu & Wilt, 1974 Tarroux, 1975 Eiden & Nichols, 1973 Armstrong et al., 1972 Hirsch, Spradling & Penman, 1974 Kruppa & Sabatini, 1977 Hirsch, Spradling & Penman, 1974 Molloy & Damell, 1973 ; Nakazato, Kopp & Edmonds, 1973 Sullivan & Roberts, 1973; Edmonds & Caramela, 1969 Mendecki, Lee & Brawerman, 1972; Jeffery & Brawerman, 1974 Baglioni, Pemberton & Delovitch, 1972 Esnault, Trapy & Van Huystee, 1975 Sagher, Edelman & Jakob, 1974 Ouellette, Kumar & Malt,. 1976 Rosenfeld et al.. 1972 Jacobsen & Zwar, 1974 Carlin, unpubl. results Mahony & Brown, 1975
TABLE
2
T4S-we
t Estimated from Table 1.
Clostridium sporenges phage Fl TT phase .I phage Bacillus amyloliquifaciens Escherichia coli Blue green algae Yeast Dictyostelium discoideum Sea urchin Silkmoth Mouse L cells Drosophilia Liver Myoblast Mosquito HeLa Rabbit blastocysts Mouse myeloma Mouse kidney Lung Lymphocyte Differentiated myoblast
Organism 0.06 0.09 0.11 0.16 0.09 0.2 0.2 0.67 2 5.6 12 15 6.5 11.6 10 13 a7 (av.) 18.3 (av.) 15 (av.) 25 18.3 (av.) 21 24 25
2.5.t 2.5t 2.5 2:; 25 25t 50 100 130 15ot 150 160 160 170.t 170 175 180t 200 212 225-f 225 225t
Half-life (h)
Poly(A) size
Greene & Korn, 1967 Taylor & Guha, 1976 Yamada, Whitaker & Nakada, 1975 Takeda & Kuwano, 1975 Brown & Coleman, 1975 Mangiarotti & Schlessinger, 1967 Leach & Carr, 1974 Fraser, 1975 Newell, Longlands & Sussman, 1971; Kessin, 1973 Nemer, Dubroff & Graham, 1975 Yund, Kafatos & Regier, 1973 Greenberg, 1972; Perry & Kelley, 1973 Lengyel & Penman, 1977 Tweedie & Pitot, 1974 Buckingham, Whalen & Gros, 1977 Spradling, Hui & Penman, 1975 Singer & Penman, 1973 Schultz, 1974 Cowan & Milstein, 1974 Ouellette & Malt, 1976 Schultz, 1973 Berger & Cooper, 1975 Buckingham, Whalen & Gros, 1977
Reference
Relationship of poly(A) length and mRNA half-life. In some instances poly(A) sizes were estimated. Estimates were obtained by comparing the tissue in question to Table 1. In some instances two half-lives were given, these were averaged (au) according to the steady state quantity of mRNA in each degradative phase
EVOLUTION
OF
POLY(A)
AND
VIRUSES
327
the poly(A) tract. The size of the poly(A) segment increases as the evolutionary level of the organism increases. Bacteriophages are found to contain the smallest poly(A) segments being only 1-5 AMP units in length. Bacteria contain the next largest poly(A) segments being only 25 AMP units in length. After bacteria the next largest poly(A) sequences are found in organelles and the least developed eucaryotes. The length of the poly(A) segment continues to increase from fungi to amphibia and insects. Mammalian cells contain the largest poly(A) segments. The large difference in poly(A) length between bacteria and bacteriophages has interesting implications in that it provides some support for the evolution of bacterial viruses before bacteria. If viruses represent degenerate bacteria as has been proposed (Green, 1935) then bacteriophages should have poly(A) segments of approximately 25 units in length. Although these poly(A) sizes indicate this order of evolution, more support is necessary since the majority of bacterial poly(A) is non-polyadenylated. This will be discussed under the evolution of viruses. The next largest poly(A) segments originate within the mitochondria of eucaryotes form the lower end of the evolutionary scale. This provides additional evidence for the theory that mitochondria originated from a primitive procaryotic cell as the mitochondrial poly(A) is of the same size as bacterial poly(A). Mitochondria isolated from Drosophila or HeLa cells have poly(A) tracts of 57 AMP units. Thus, poly(A) in mitochondria is found to be evolving to larger sizes. Chloroplast poly(A) also is of a small size resembling procaryotic poly(A). Caulobacter crescentus represents an interesting evolutionary case. As a bacteria, it has been shown to have the ability to differentiate (Shapiro, 1976). It has a poly(A) segment which ranges from 15, within the size of bacteria, to 50 bases, approaching the size of the smallest eucaryotic poly(A) segments. Thus caulobacter may possibly represent an intermediate between bacteria and the lowest eucaryotes in evolution. In this respect, caulobacter could possibly be used as a model system to study the transition of procaryotes to eucaryotes. Another aspect of mRNA which has evolved in a manner similar to that of poly(A) is the stability of mRNA (Table 2). A plot of the log of average poly(A) size and the log of average mRNA half-life for different organisms results in a linear relationship (Fig. 1). This correlation suggests that the role of poly(A) is to stabilize mRNA. An interesting point demonstrated in Fig. 1 is that a poly(A) segment of 21 is required before stabilization of mRNA occurs. This agrees with the half-life studies by Nude& Soreq, & Littauer (1976) in which globin mRNA containing various poly(A) lengths was injected into frog oocytes. In order to confer any stability to globin
328
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-+----
CARLIN
0 0
I
I
I
2 Log A
FIG. 1. Relationship of the log of newly synthesized poly (A) size and the bg of mRNA half-life. Each point represents the average mRNA half-life and the average newly synthesized poly (A) size for a single organism. Points were obtained from Table 2. Open circles represent points with estimated poly (A) values and were not used in the least squares plot. Correlational coefficient = 0.98, standard error = ~tO.34 T/2.
mRNA a poly(A) segment of at least 21 AMP units had to be attached to the mRNA. However unlike the oocyte studies, stability continued to increase after poly(A) sizes became greater than 32. Hieter, LeGendre & Levy (1976) found that the addition of poly(A) to E. coli 5 S RNA resulted in an increased stability of the RNA in vitro. The increase in stability was proportional to the length of the poly(A) segment. Although these studies suggest poly(A) and mRNA stability are related, the present investigation represents the first in vivo evidence that poly(A) regulates mRNA stability in which (1) poly(A) or mRNA had not been manipulated before the experiment, (2) mRNA half-lives were obtained in vivo from intact organisms thus preserving the normal degradative mechanisms for the mRNA and (3) the relationship was demonstrated among a diverse group of organisms. A relationship has also been shown between mRNA stability and cell generation time (Sensky, Haines & Rees, 1975). A parallel increase of cell generation time could account for increased mRNA half-life. A comparison of mRNA half-life and cell generation time reveals that increased cell generation time is not the primary reason for increased mRNA half-life. In E. coli, the half-life of mRNA represents 3-6% of the cell doubling time
EVOLUTION
OF
POLY(A)
AND
VIRUSES
329
(Mangiarotti & Schlessinger, 1967) whereas in yeast the half-life of mRNA is lo-15 % of the cell cycle (Peterson, McLaughlin, & Nierlich, 1976) and in mouse L cells the mRNA half-life represents 70 % or more of the cell generation time (Greenberg, 1972). Thus the increase in mRNA stability cannot be attributed only to changes in cell generation time. However, variations in cell generation time could account for deviations from perfect linearity in Fig. 1. Another observation is that cancer cells have smaller poly(A) sizes than differentiated tissue. During differentiation of erythrocytes and neuroblastoma cells, poly(A) polymerase has been shown to be stimulated (Lee, 1976; Simantov & Sachs, 1975). In myoblasts, mRNA half-lives were found to increase from 10 h to 25 h as the cell underwent differentiation (Buckingham, Whalen & Gros, 1977). Thus the increase in mRNA stability and poly(A) size in differentiated cells also support a role of poly(A) in stabilizing mRNA. Provided there is a direct relationship between poly(A) size and mRNA stability as is implicated in Fig. 1 then certain predictions can be made. Among these are that the curve shown in Fig. 1 should be applicable to specific size classes of poly(A) +mRNA and specific mRNA within organisms. The test best proving the direct relationship of poly(A) and stability would be the prediction of specific mRNA half-lives from poly(A) size or vice versa. Since certain mRNA molecules could be regulated by mechanisms different than by poly(A) alone certain restrictions must be placed on predictions concerning specific mRNA species. One restriction is that caution must be used in applying this relationship to terminally differentiated cells. An example of this is globin mRNA. It has a newly synthesized poly(A) size of 150 nucleotides (Pemberton & Baglioni, 1972; Merkel, Kwan, & Lingrel, 1975) and its predicted half-life, by Fig. 1, is lo+ 5 h. In terminally differentiated reticulocytes, however, the half-life is 24 h (Lodish & Small, 1976). In this instance, where the differentiated cells have produced one species of mRNA and have, also, undergone other processes, the decay of message may be regulated differently than by poly(A) alone. One possible explanation for the stability of globin mRNA is that RNases within the reticulocyte are greatly reduced. Another explanation is that since the production of globin in these cells is of utmost importance, a message specific protein could be retarding its degradation. In cells which are induced to produce globin mRNA but which are undifferentiated the predicted halflife of the mRNA should be between 5 and 15 h. This is found in Friend erythroleukemia cells (Aviv, Voloch, Bastos, & Levy, 1976), a less differentiated cell, in which the half-life of the message is 16-17 h approaching the upper limit of the predicted half-life. Another restriction on predicting
330
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CARLIN
half-lives concerns masked mRNA whose degradation is prevented by protein. Therefore mRNA should be involved in translation for the relationship in Fig. 1 to be applicable. The following equations described the relationship between mRNA half-life and poly(A) size: Predicted range of mRNA half-life = T/2) T/2(0.5) where : T/2
=
102.33
log(A)-4.08
A = poly(A) size in AMP units. This equation should be able to predict half-lives and poly(A) segment sizes provided the following conditions are met: (1) the cells are not deficient in ribonucleases, (2) the mRNA is not masked, and (3) the mRNA is not the predominant species requiring specialized degradative controls involving proteins. Among some mRNA predictions resulting from Fig. 1 are chick vitellogenin mRNA (poly(A)-220, Jost & Pehling, 1976) half-life : 23.5 + 12 h and ovalbumin mRNA (poly(A)-240, Rhoads, 1975) half-life: 29 + 14 h. In agreement with one of these predictions, Palmiter (1973) has reported the half-lie of ovalbumin mRNA to be 24 h. Also any other mRNA with a known poly(A) size should have a half-life within the range predicted by the above equation as long as the previously discussed restrictions are used. The mRNA for the immunogIobin light chain (half-life: 12-14 h, Cowan & Milstein, 1974) would be predicted to have a poly(A) size of 170$- 40. Also mRNA from seeds would be expected to have very long poly(A) segments since they contain mRNA with very long half-lives (Payne, 1976). The mRNA from seeds would not match Fig. 1 as other factors such as extremely low metabolism are present. In considering the stabilizing effect of poly(A) on mRNA, how can the stability of non-polyadenylated mRNA be accounted for. In HeLa and L cells, one third of all mRNA had been reported to lack a poly(A) segment (Greenberg, 1976; Milcarek, Prince, & Penman, 1974). The degradation of non-polyadenylated mRNA appears to follow that of polyadenylated mRNA (Milcarek et al., 1974; Nemer, Dubroff & Graham, 1975). Studies by Perry & Kelley (1973) showed that poly(A) containing mRNA degrades by a stochastic process while histone mRNA lacking a poly(A) segment degrades by an age dependent decay or zero ordered turnover. This suggests that the mechanism of decay for the two classes of mRNA are very different. The normal half-life of histone mRNA in exponentially growing cells is 6 h (Perry & Kelley, 1973) but when DNA synthesis is disrupted the half-life becomes 10-30 min (Gallwitz, 1975; Stahl & Gallwitz, 1977). This halflife is very close to that predicted for mRNA lacking a poly(A) segment.
EVOLUTION
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AND
VIRUSES
331
Since histone mRNA represents 40 % of the total non-polyadenylated mRNA in L cells (Greenberg, 1976) this mRNA could possibly be a good representative of the entire class of non-polyadenylated mRNA. The studies of histone mRNA suggest that possible general characteristics of the nonpolyadenylated mRNA are (1) degradation occurs by a mechanism different from polyadenylated mRNA and is coupled to DNA synthesis, (2) if DNA synthesis ends or is disrupted the mRNA degrades by the same mechanism as polyadenylated mRNA and has a very short half-life, and (3) as a corollary, mRNA lacking poly(A) are generally specific to the S phase of the cell cycle. However, more studies are necessary on the nonpolyadenylated nonhistone mRNA to determine if they have the same characteristics as the histone mRNA. The relationship of poly(A) length and viruses is listed in Table 3. Since poly(A) length is related to the evolutionary status of non-viral organisms the possibility exists that the length of poly(A) segments in viruses may be used to help elucidate the evolution of viruses. A comparison of different families of RNA viruses reveals this to be the case. The five classes of RNA viruses which will be discussed are used since the poly(A) size has been determined for more than one member of each family. The following values represent the average poly(A) size of each family: Picornaviridae (50), Togaviridae (68), Paramyxoviridae & Orthomyxoviridae (113), and Retroviridae (175). A comparison of these families reveal an increase in “evolutionary complexity” as poly(A) size increases. Poly(A) is not related to the structural complexity of viruses but to the ability of the viruses to interact with more evolved organisms (evolutionary complexity). As an example, although SV40 is much simpler in structure than some bacteriophage, it has the ability to become integrated and function in the eucaryotic genome whereas the bacteriophage cannot. The family picornaviridae contains the simplest RNA viruses. The viruses are characterized by small icosahedral particles consisting of usually four proteins and one molecule of RNA. Replication occurs entirely in the cytoplasm. The family togaviridae contains the next most evolved viruses. Like the picornaviridae the viruses are icosahedral and contain one single stranded RNA molecule. After replication in the cytoplasm, however, the viruses mature by budding through the plasma membrane. The next two groups, orthomyxoviridae and paramyxoviridae, are surrounded by a membrane. They also contain RNA polymerase and neuriminidase within the virion, have a helical nucleoprotein core, and some replicate in the nucleus. The last family, retroviridae, are the most complex. They contain two or more molecules of RNA, one molecule of reverse transcriptase, many proteins, and membranes. Replication occurs entirely in the nucleus.
TABLE 3
Parvoviridae Adenoviridae
Tobamovirus Picornaviridae Picornaviridae Picornaviridae Picornaviridae Togaviridae Picornaviridae Picomaviridae Togaviridae Covirus Paramyxoviridae Togaviridae Picornaviridae Rhabdoviridae Poxviridae Paramyxoviridae Orthomyxoviridae Orthomyxoviridae Retroviridae Herpetoviridae Retroviridae Retroviridae Papavaviridae Retroviridae Comovirus Retroviridae
Family RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA DNA RNA RNA RNA RNA DNA RNA RNA DNA RNA RNA RNA RNA DNA DNA
Nucleic acid
TDerived from electrophoretic and sucrose gradient S values. $.Oncogenic.
Tobacco mosaic Nodamura Encephalomyocarditis Rhinovirus Mengovirus Sindbis Columbia SK Foot & Mouth Eastern equine encephalitis Bean pod mottle Sendai Semliki forest Polio Vesicular stomatitis Vaccinia Newcastle disease Influenza Fowl Plague Murine sarcomat Herpes simplex3 Murine leukemia sarcoma$ Mouse leukemia2 sv 401 Rous sarcomat Cowpea mosaic Visnal: Mason Pfizer agent$ Adeno associated AdenovirusS
Virus
;i 75-125 100-150 120-130 SO-200 loo-150 100-200 16Ot 1707 170 150-200 170-200 200 200 200 200 150-250
80-t
4: 38-48 SO-55 30-80 30-SO? 60-80 70 55-95’r
0
Poly(A) size
Fraser, 1973 Neuman & Brown, 1976 Frisby, Smith, Jeffers & Porter, 1976 Nair & Panicala, 1976 Marshall & Arlinghaus, 1976 Deborde & Leibowitz, 1976; Eaton & Faulkner, 1972 Johnston & Rose, 1972 Chatterjee, Bachrach & Polatnik, 1976 Armstrong et al., 1972 Semancik, 1974 Pridgen & Kingsbury, 1972 Clegg & Kennedy, 1974 Armstrong et al., 1972; Yogo & Wimmer, 1972 Soria & Huang, 1973; Rose & Knipe, 1975 Nevins & Joklik, 1975; Kates & Beeson, 1970 Weiss & Bratt, 1974 Etkind & Krug, 1974 Ghendon & Blagovesbienskaya, 1975 Green & Cartas, 1972 Bachenheimer & Roizman, 1972 Ross, Tronick & Scholnick, 1972 Lai & Duesberg, 1972 Weinberg, Ben-Ishai & Newbold, 1972 Wang & Duesberg, 1974; Lai & Duesberg, 1972 Manna & Bruening, 1973 Gillespie et al., 1973 Gillespie et al., 1973 Carter, 1976 Philipson et al., 1971
References
Relationship of poly(A) and viruses. Average poly(A) sizesfor difSerent viruses have been listed in increasing order
EVOLUTION
OF POLY(A)
AND
333
VIRUSES
The parallel increase in poly(A) size and “evolutionary complexity” in eucaryotic viruses suggests two possible modes of evolution of viruses. The fitst possible mode for the evolution of viruses would involve the evolution of viruses parallel with eucaryotic cells. The poly(A) size of the simplest viruses, picornaviridae, have an average poly(A) size of about 50 AMP units which is approximately the poly(A) of the primitive eucaryotes. Since there are smaller poly(A) sizes within the family, the eucaryotic viruses could have evolved from procaryotic cells or procaryotic RNA phages. The evolution of the viruses to more complex forms would then occur as eucaryotic cells evolved to more and more complex forms. The second possible mode of evolution for viruses could be that viruses could have evolved at different times. As eucaryotic cells became more evolved, viruses which formed from the cell machinery (protovirus theory, Temin, 1974) would have poly(A) sizes approximately equal to the cell at that stage of evolution. A problem with this theory is that viruses, after spontaneously forming from a eucaryotic cell must cease to evolve at a significant rate. Another relationship which is apparent in Table 3 is that animal viruses with poly(A) segments of about 200 are generally oncogenic. If poly(A) size does represent an evolutionary pattern in viruses, it would indicate oncogemc viruses are the most recent to develop. One reason these viruses would tend to have poly(A) segments of this length would result from the ability of these viruses to produce mRNA using the cells machinery. On the basis of poly(A) no distinction can be made on whether oncogenic viruses originated according to the protovirus theory of Temin or by continuous chain of evolution. By using the poly(A) size of viruses the following evolutionary pattern emerges (Fig. 2). The Crst organism to have evolved would be a virus-like
I 0
I 50
I 100
I
1
150
200
Poly (A) size
FIG. 2. Proposed evolution of viruses. Arrows indicate the direction of evolution. Viruses and eucaryotic cells are proposed to be evolving in parallel. Interactions between the viruses and cells are assumed to occur at all times. Dotted lines indicate possible pathways. The starred pathway indicates the evolution of RNA phages from nonpolyadenylated bacterial mRNA, not a reverse evolutionary process.
334
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structure. This is likely since a nucleic acid-protein complex would be easier to form from prebiotic materials than would a completely metabolically active cell. The previrus could “parasitize” the prebiotic ocean in order to replicate and evolve. The previrus would slowly evolve into a procaryotic cell. Later the previrus would evolve into DNA bacteriophages. These bacteriophages would “parasitize” the bacteria as the prebiotic ocean became depleted of organic molecules. RNA bacteriophages, because of their mRNA characteristics, probably evolved from bacterial mRNA. This would also be likely since RNA would be very unstable in the prebiotic ocean. As procaryotic cells developed into eucaryotic cells the resulting gross changes within the cells would prevent the infection of the cells by bacteriophages. Bacteria would then begin to invade eucaryotic cells. Some of these invasions might have resulted in the formation of mitochondria and chloroplasts. In other cases a portion of the bacterial cell machinery could have evolved into primitive eucaryotic viruses. At this stage, the evolution of viruses from procaryotes is more likely than the formation of protoviruses from eucaryotic cells since the replication of simple viruses occur in the cytoplasm. This indicates that the nuclear cell machinery of the developing eucaryotic cell is incompatible with viral replicative mechanisms, as would be the case for a procaryotic replicative mechanism in a eucaryotic cell. An observation from Table 3, which is directly applicable to the evolution of viruses is the difference in poly(A) size between RNA and DNA viruses. DNA viruses (average poly(A) size-171) do not appear until after a poly(A) size of greater than 100 is reached whereas RNA viruses with poly(A) sizes less than 100 are abundant. This would indicate that RNA animal viruses developed first. In the case of the formatiion of the first eucaryotic virus from bacterial cell machinery, it would seem reasonable that RNA would be involved in viral evolution instead of bacterial DNA. Bacterial DNA would have no relationship with the DNA replicating machinery of the cell whereas bacterial mRNA is similar to eucaryotic mRNA. Thus by the addition of a single protein, a RNA directed RNA polymerase, a mechanism for the replication of viral RNA outside the nucleus would be present. Also consistent with the formation of animal viruses from bacterial mRNA is a pathway involving RNA phages. This would be essentially the same pathway except the formation of RNA viruses would occur first. Thus a bacterial mRNA could form a RNA bacteriophage which would continue to evolve and later invade early eucaryotes. The RNA viruses would continue to evolve in a parallel manner as the eucaryotic cells. During evolution, mechanisms probably developed in cells which accelerated evolution. One of these mechanisms might include the use of reverse transcriptase (Temin, 1974). When RNA viruses infected these
EVOLUTION
OF
POLY(A)
AND
335
VIRUSES
cells, DNA copies suitable for manipulation in the cell nucleus would be generated. Also some DNA viruses could have been formed from these transcripts. Table 2 indicates that DNA viruses and RNA viruses containing reverse transcriptase have approximately the same average poly(A) size (DNA viruses-171, Retroviridae-175) indicating their evolution at approximately the same time. The RNA viruses, after having the DNA copies incorporated into the genome, could detach from the genome with parts of the eucaryotic genome. Eventually the viruses would become integrated with some control elements and thus aquire the ability to alter cells (oncogenic viruses). According to natural selection a virus which could induce a cell to increase its rate of division would be more likely to survive since more copies of itself would be present without destroying the host. If this theory is correct more refined measurements of poly(A) size could be used to determine at what point in evolution reverse transcriptase developed. A comparison of poly(A) and evolution have produced the following general conclusions: (1) poly(A) h as evolved in length ; (2) poly(A) is characteristic of many different classes of organisms ; (3) mitochondrial poly(A) is evolving to longer lengths; (4) poly(A) appears to regulate the stability of mRNA ; and (5) poly(A) size can be used to study the evolutionary order of viruses. As further poly(A) size determinations are made these tables can be refined and thus portray a more precise picture of what has been proposed in this paper. One of the most fruitful areas for poly(A) determinations is in the field of eucaryotic viruses. As more poly(A) sizes are determined a better evolutionary relationship can be drawn between other families and orders of viruses. Also the evolutionary relationships between plant, protist, and animal viruses can be determined more precisely. Since poly(A) sizes tend to remain the same for individual families, i.e. picornaviridae, poly(A) size could be used to help classify viruses. Thus the relationship between poly(A) size and evolution offers a new taxonomic method to study the evolution of viruses. I wish to thank Dr A. P. Kimball for valuable discussions and editorial assistance. Thanks are also due to Dr W. R. Jeffery and Mr D. S. Adams for helpful suggestions. REFERENCES ADAMS, D. S. & JEWERY, W. R. (1977). Fed. Proc. 36,2617. ARMSTRONG, J. A., EDMONDS, M., NAKAZATO, H., PHILLIPS, B. A. & VAUGHAN, (1972). Science 176, 526. AVN, H., VOLOCH, Z., BASTOS, R. & LEVY, S. (1976). Cell 8,495. BACHENHEIMER, S. L. & ROIZMAN, B. (1972). J. Virol. 10, 875. BAGLIONI, C., PEMBERTON, R. & DELQVITCH, T. (1972).FEBS Lett. 26, 320. BERGER, S. L. & COOPER, H. L. (1975).Proc. mtn. Acad. Sci., U.S.A. 72, 3873.
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Note added in proof The log of average poly(A) size for different organisms has been recently shown to have evolved in a linear manner with the time of origin for the organisms (Carlin, in prep.).
The Poly(A) Segment of mRNA: (1) Evolution and Function and (2) The Evolution of Viruses RICHARD K. CARLIN University
of Houston, Department of BiophJjsical Sciences, Houston, Texas 77004, U.S.A.
(Received 25 February
1977, and in revisedform
22 August 1977)
The poly(A) segment of mRNA has been shown to be related to evolution. The poly(A) segment increases in length as the complexity of the organism increases. The shortest poly(A) size (1-5 AMP units) exists on mRNA from bacteriophages. The largest poly(A) segments (200-250 AMP units) exist in highly differentiated tissue. The log of poly(A) size and the log of mRNA half-life for different organisms was found to be related in a linear manner. Predictions of specific mRNA half-lives and poly(A) sizes were made to help show a direct relationship. This relationship was also valid for histone poly(A) minus mRNA, provided DNA synthesis is interrupted. Poly(A) segments from viruses reflect the evolutionary status of the viruses. On the basis of viral poly(A) sizes the origin and evolution of viruses is proposed. Important points of this theory are (1) a type of previrus preceded procaryotic cells, (2) these previruses developed into procaryotic cells and DNA bacteriophages, (3) the first eucaryotic viruses contained RNA and originated from procaryotic mRNA, (4) DNA viruses may have developed from RNA viruses as reverse transcriptase developed, and (5) continued evolution resulted in the development of oncogenic viruses.
1. The Poly(A) Segment of mRNA: (1) Evolution and Function and (2) The Evolution of Virnses Poly(A) has been found to be a major component of mRNA (Edmonds & Caramela, 1969; Lee, Mendecki, & Brawerman, 1971). This segment is attached to the 3’ end of mRNA and ranges in size up to 250 AMP units long (Brawerman, 1974). The existence of poly(A) tracts has prompted a great deal of speculation relating to the precise function of these polynucleotide sequences. Possible functions which have been proposed are that poly(A) may be involved in the transport, translation, or stability of mRNA. One method which might aid in the elucidation of poly(A) function was to study its evolution. Table 1 shows the relationship of poly(A) size and the source from which it was obtained. A definite pattern is formed on the basis of the length of 323
0022-.5193/78/0406-0323$.0200/O
0 1978Academic Press Inc. (London) Ltd.
TABLE 1
viridae
Drosophilia & HeLa Achlya ambisexualis, fungus Neurospora crassa, fungus Naegleria gruberi, amebae Cotton
Mitochondria,
Blastocladiella emersonii, fungus Rhizopus stolonifer, fungus Trichoderma viridae, fungus
Chloroplast, maize Chlorella, green algae Yeast Phaseolus areus, mung bean
Caulobacter
Trichoderma
yeast
crescentus
coli
Mitochondria, Mitochondria,
Escherichia
n phage T7 phage 480 phage
Organism
2 50-100 80 75-100
50-60
50 5ot 50 50
:2+
15-50
20-30
25 20-25
2-5
l-5
1-5
Poly(A) size Rosenberg, Weissman & DeCrombrugghe, 1975 Kramer, Rosenberg & Steitz, 1974 Pieczenik, Barrel1 & Gefter, 1972 Nakazato, Venkatesan & Edmonds, 1975 Rosen & Edelman, 1976 Hendler ef al., 1975 Ohta, Sanders & Newton, 1975 Haff & Bogorad, 1976 Scragg & Thurster, 1975 McLaughlin et al., 1973 Higgins, Mercer & Goodwin, 1973 Jaworski, 1976 Freer, Mayama & Etten, 1977 Rosen & Edelman, 1976; Rosen, Edelman & Gallum, 1975 Hirsch & Penman, 1973 Silver & Horgen, 1974 Mirkes & McCalley, 1976 Walsh, Nakazato, Hickey & Edmonds, 1976 Van De Walle, 1973
listed in increasing size
Relationship of poly(A) and evolution. Average newly synthesized poly(A) lengths for dz@erentorganisms have been
pyriformis
gracilis
7 Derived from electrophoretic
Mouse kidney Lymphocyte Barley, aleuron tissue Differentiated neuroblastoma Rabbit brain
Euglena
100 125 120-150 SO-200 150 140-l 60t 15@-160t 160t 14&180 170 130-200 145-210 170-220 200 200 150-200 200-225 225 200-250 200-250 200-250
K120t
90
and sucrose gradient S values.
Pieris brassicae, wing imaginal disk Mouse L cells Chick embryonic cells Drosophila Liver Mosquito HeLa cells Ehrlich ascites Sarcoma 180 Myeloma yiciafaba, meristematic root tissue
Sea urchin
Dictyostelium discoidem, cellular slime mold Physarum polycephalum, unicellular slime mold Xenopus laevis, embryonic cells
Maize Rice, callus tissue
Tetrahymena
Rodriquez-Pousada & Hayes, 1976 Haff & Bogorad, 1976 Manahan, App & Still, 1973 Firtel, Jacobson & Lodish, 1972; Sheiness & Damell, 1973 Adams & Jeffery, 1977 Dina, Meza & Crippa, 1974; Sagata, Shinokawa & Yamana, 1976 Slater, Slater & Gillespie, 1972; Wu & Wilt, 1974 Tarroux, 1975 Eiden & Nichols, 1973 Armstrong et al., 1972 Hirsch, Spradling & Penman, 1974 Kruppa & Sabatini, 1977 Hirsch, Spradling & Penman, 1974 Molloy & Damell, 1973 ; Nakazato, Kopp & Edmonds, 1973 Sullivan & Roberts, 1973; Edmonds & Caramela, 1969 Mendecki, Lee & Brawerman, 1972; Jeffery & Brawerman, 1974 Baglioni, Pemberton & Delovitch, 1972 Esnault, Trapy & Van Huystee, 1975 Sagher, Edelman & Jakob, 1974 Ouellette, Kumar & Malt,. 1976 Rosenfeld et al.. 1972 Jacobsen & Zwar, 1974 Carlin, unpubl. results Mahony & Brown, 1975
TABLE
2
T4S-we
t Estimated from Table 1.
Clostridium sporenges phage Fl TT phase .I phage Bacillus amyloliquifaciens Escherichia coli Blue green algae Yeast Dictyostelium discoideum Sea urchin Silkmoth Mouse L cells Drosophilia Liver Myoblast Mosquito HeLa Rabbit blastocysts Mouse myeloma Mouse kidney Lung Lymphocyte Differentiated myoblast
Organism 0.06 0.09 0.11 0.16 0.09 0.2 0.2 0.67 2 5.6 12 15 6.5 11.6 10 13 a7 (av.) 18.3 (av.) 15 (av.) 25 18.3 (av.) 21 24 25
2.5.t 2.5t 2.5 2:; 25 25t 50 100 130 15ot 150 160 160 170.t 170 175 180t 200 212 225-f 225 225t
Half-life (h)
Poly(A) size
Greene & Korn, 1967 Taylor & Guha, 1976 Yamada, Whitaker & Nakada, 1975 Takeda & Kuwano, 1975 Brown & Coleman, 1975 Mangiarotti & Schlessinger, 1967 Leach & Carr, 1974 Fraser, 1975 Newell, Longlands & Sussman, 1971; Kessin, 1973 Nemer, Dubroff & Graham, 1975 Yund, Kafatos & Regier, 1973 Greenberg, 1972; Perry & Kelley, 1973 Lengyel & Penman, 1977 Tweedie & Pitot, 1974 Buckingham, Whalen & Gros, 1977 Spradling, Hui & Penman, 1975 Singer & Penman, 1973 Schultz, 1974 Cowan & Milstein, 1974 Ouellette & Malt, 1976 Schultz, 1973 Berger & Cooper, 1975 Buckingham, Whalen & Gros, 1977
Reference
Relationship of poly(A) length and mRNA half-life. In some instances poly(A) sizes were estimated. Estimates were obtained by comparing the tissue in question to Table 1. In some instances two half-lives were given, these were averaged (au) according to the steady state quantity of mRNA in each degradative phase
EVOLUTION
OF
POLY(A)
AND
VIRUSES
327
the poly(A) tract. The size of the poly(A) segment increases as the evolutionary level of the organism increases. Bacteriophages are found to contain the smallest poly(A) segments being only 1-5 AMP units in length. Bacteria contain the next largest poly(A) segments being only 25 AMP units in length. After bacteria the next largest poly(A) sequences are found in organelles and the least developed eucaryotes. The length of the poly(A) segment continues to increase from fungi to amphibia and insects. Mammalian cells contain the largest poly(A) segments. The large difference in poly(A) length between bacteria and bacteriophages has interesting implications in that it provides some support for the evolution of bacterial viruses before bacteria. If viruses represent degenerate bacteria as has been proposed (Green, 1935) then bacteriophages should have poly(A) segments of approximately 25 units in length. Although these poly(A) sizes indicate this order of evolution, more support is necessary since the majority of bacterial poly(A) is non-polyadenylated. This will be discussed under the evolution of viruses. The next largest poly(A) segments originate within the mitochondria of eucaryotes form the lower end of the evolutionary scale. This provides additional evidence for the theory that mitochondria originated from a primitive procaryotic cell as the mitochondrial poly(A) is of the same size as bacterial poly(A). Mitochondria isolated from Drosophila or HeLa cells have poly(A) tracts of 57 AMP units. Thus, poly(A) in mitochondria is found to be evolving to larger sizes. Chloroplast poly(A) also is of a small size resembling procaryotic poly(A). Caulobacter crescentus represents an interesting evolutionary case. As a bacteria, it has been shown to have the ability to differentiate (Shapiro, 1976). It has a poly(A) segment which ranges from 15, within the size of bacteria, to 50 bases, approaching the size of the smallest eucaryotic poly(A) segments. Thus caulobacter may possibly represent an intermediate between bacteria and the lowest eucaryotes in evolution. In this respect, caulobacter could possibly be used as a model system to study the transition of procaryotes to eucaryotes. Another aspect of mRNA which has evolved in a manner similar to that of poly(A) is the stability of mRNA (Table 2). A plot of the log of average poly(A) size and the log of average mRNA half-life for different organisms results in a linear relationship (Fig. 1). This correlation suggests that the role of poly(A) is to stabilize mRNA. An interesting point demonstrated in Fig. 1 is that a poly(A) segment of 21 is required before stabilization of mRNA occurs. This agrees with the half-life studies by Nude& Soreq, & Littauer (1976) in which globin mRNA containing various poly(A) lengths was injected into frog oocytes. In order to confer any stability to globin
328
R.
K.
-+----
CARLIN
0 0
I
I
I
2 Log A
FIG. 1. Relationship of the log of newly synthesized poly (A) size and the bg of mRNA half-life. Each point represents the average mRNA half-life and the average newly synthesized poly (A) size for a single organism. Points were obtained from Table 2. Open circles represent points with estimated poly (A) values and were not used in the least squares plot. Correlational coefficient = 0.98, standard error = ~tO.34 T/2.
mRNA a poly(A) segment of at least 21 AMP units had to be attached to the mRNA. However unlike the oocyte studies, stability continued to increase after poly(A) sizes became greater than 32. Hieter, LeGendre & Levy (1976) found that the addition of poly(A) to E. coli 5 S RNA resulted in an increased stability of the RNA in vitro. The increase in stability was proportional to the length of the poly(A) segment. Although these studies suggest poly(A) and mRNA stability are related, the present investigation represents the first in vivo evidence that poly(A) regulates mRNA stability in which (1) poly(A) or mRNA had not been manipulated before the experiment, (2) mRNA half-lives were obtained in vivo from intact organisms thus preserving the normal degradative mechanisms for the mRNA and (3) the relationship was demonstrated among a diverse group of organisms. A relationship has also been shown between mRNA stability and cell generation time (Sensky, Haines & Rees, 1975). A parallel increase of cell generation time could account for increased mRNA half-life. A comparison of mRNA half-life and cell generation time reveals that increased cell generation time is not the primary reason for increased mRNA half-life. In E. coli, the half-life of mRNA represents 3-6% of the cell doubling time
EVOLUTION
OF
POLY(A)
AND
VIRUSES
329
(Mangiarotti & Schlessinger, 1967) whereas in yeast the half-life of mRNA is lo-15 % of the cell cycle (Peterson, McLaughlin, & Nierlich, 1976) and in mouse L cells the mRNA half-life represents 70 % or more of the cell generation time (Greenberg, 1972). Thus the increase in mRNA stability cannot be attributed only to changes in cell generation time. However, variations in cell generation time could account for deviations from perfect linearity in Fig. 1. Another observation is that cancer cells have smaller poly(A) sizes than differentiated tissue. During differentiation of erythrocytes and neuroblastoma cells, poly(A) polymerase has been shown to be stimulated (Lee, 1976; Simantov & Sachs, 1975). In myoblasts, mRNA half-lives were found to increase from 10 h to 25 h as the cell underwent differentiation (Buckingham, Whalen & Gros, 1977). Thus the increase in mRNA stability and poly(A) size in differentiated cells also support a role of poly(A) in stabilizing mRNA. Provided there is a direct relationship between poly(A) size and mRNA stability as is implicated in Fig. 1 then certain predictions can be made. Among these are that the curve shown in Fig. 1 should be applicable to specific size classes of poly(A) +mRNA and specific mRNA within organisms. The test best proving the direct relationship of poly(A) and stability would be the prediction of specific mRNA half-lives from poly(A) size or vice versa. Since certain mRNA molecules could be regulated by mechanisms different than by poly(A) alone certain restrictions must be placed on predictions concerning specific mRNA species. One restriction is that caution must be used in applying this relationship to terminally differentiated cells. An example of this is globin mRNA. It has a newly synthesized poly(A) size of 150 nucleotides (Pemberton & Baglioni, 1972; Merkel, Kwan, & Lingrel, 1975) and its predicted half-life, by Fig. 1, is lo+ 5 h. In terminally differentiated reticulocytes, however, the half-life is 24 h (Lodish & Small, 1976). In this instance, where the differentiated cells have produced one species of mRNA and have, also, undergone other processes, the decay of message may be regulated differently than by poly(A) alone. One possible explanation for the stability of globin mRNA is that RNases within the reticulocyte are greatly reduced. Another explanation is that since the production of globin in these cells is of utmost importance, a message specific protein could be retarding its degradation. In cells which are induced to produce globin mRNA but which are undifferentiated the predicted halflife of the mRNA should be between 5 and 15 h. This is found in Friend erythroleukemia cells (Aviv, Voloch, Bastos, & Levy, 1976), a less differentiated cell, in which the half-life of the message is 16-17 h approaching the upper limit of the predicted half-life. Another restriction on predicting
330
R.
K.
CARLIN
half-lives concerns masked mRNA whose degradation is prevented by protein. Therefore mRNA should be involved in translation for the relationship in Fig. 1 to be applicable. The following equations described the relationship between mRNA half-life and poly(A) size: Predicted range of mRNA half-life = T/2) T/2(0.5) where : T/2
=
102.33
log(A)-4.08
A = poly(A) size in AMP units. This equation should be able to predict half-lives and poly(A) segment sizes provided the following conditions are met: (1) the cells are not deficient in ribonucleases, (2) the mRNA is not masked, and (3) the mRNA is not the predominant species requiring specialized degradative controls involving proteins. Among some mRNA predictions resulting from Fig. 1 are chick vitellogenin mRNA (poly(A)-220, Jost & Pehling, 1976) half-life : 23.5 + 12 h and ovalbumin mRNA (poly(A)-240, Rhoads, 1975) half-life: 29 + 14 h. In agreement with one of these predictions, Palmiter (1973) has reported the half-lie of ovalbumin mRNA to be 24 h. Also any other mRNA with a known poly(A) size should have a half-life within the range predicted by the above equation as long as the previously discussed restrictions are used. The mRNA for the immunogIobin light chain (half-life: 12-14 h, Cowan & Milstein, 1974) would be predicted to have a poly(A) size of 170$- 40. Also mRNA from seeds would be expected to have very long poly(A) segments since they contain mRNA with very long half-lives (Payne, 1976). The mRNA from seeds would not match Fig. 1 as other factors such as extremely low metabolism are present. In considering the stabilizing effect of poly(A) on mRNA, how can the stability of non-polyadenylated mRNA be accounted for. In HeLa and L cells, one third of all mRNA had been reported to lack a poly(A) segment (Greenberg, 1976; Milcarek, Prince, & Penman, 1974). The degradation of non-polyadenylated mRNA appears to follow that of polyadenylated mRNA (Milcarek et al., 1974; Nemer, Dubroff & Graham, 1975). Studies by Perry & Kelley (1973) showed that poly(A) containing mRNA degrades by a stochastic process while histone mRNA lacking a poly(A) segment degrades by an age dependent decay or zero ordered turnover. This suggests that the mechanism of decay for the two classes of mRNA are very different. The normal half-life of histone mRNA in exponentially growing cells is 6 h (Perry & Kelley, 1973) but when DNA synthesis is disrupted the half-life becomes 10-30 min (Gallwitz, 1975; Stahl & Gallwitz, 1977). This halflife is very close to that predicted for mRNA lacking a poly(A) segment.
EVOLUTION
OF
POLY(A)
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Since histone mRNA represents 40 % of the total non-polyadenylated mRNA in L cells (Greenberg, 1976) this mRNA could possibly be a good representative of the entire class of non-polyadenylated mRNA. The studies of histone mRNA suggest that possible general characteristics of the nonpolyadenylated mRNA are (1) degradation occurs by a mechanism different from polyadenylated mRNA and is coupled to DNA synthesis, (2) if DNA synthesis ends or is disrupted the mRNA degrades by the same mechanism as polyadenylated mRNA and has a very short half-life, and (3) as a corollary, mRNA lacking poly(A) are generally specific to the S phase of the cell cycle. However, more studies are necessary on the nonpolyadenylated nonhistone mRNA to determine if they have the same characteristics as the histone mRNA. The relationship of poly(A) length and viruses is listed in Table 3. Since poly(A) length is related to the evolutionary status of non-viral organisms the possibility exists that the length of poly(A) segments in viruses may be used to help elucidate the evolution of viruses. A comparison of different families of RNA viruses reveals this to be the case. The five classes of RNA viruses which will be discussed are used since the poly(A) size has been determined for more than one member of each family. The following values represent the average poly(A) size of each family: Picornaviridae (50), Togaviridae (68), Paramyxoviridae & Orthomyxoviridae (113), and Retroviridae (175). A comparison of these families reveal an increase in “evolutionary complexity” as poly(A) size increases. Poly(A) is not related to the structural complexity of viruses but to the ability of the viruses to interact with more evolved organisms (evolutionary complexity). As an example, although SV40 is much simpler in structure than some bacteriophage, it has the ability to become integrated and function in the eucaryotic genome whereas the bacteriophage cannot. The family picornaviridae contains the simplest RNA viruses. The viruses are characterized by small icosahedral particles consisting of usually four proteins and one molecule of RNA. Replication occurs entirely in the cytoplasm. The family togaviridae contains the next most evolved viruses. Like the picornaviridae the viruses are icosahedral and contain one single stranded RNA molecule. After replication in the cytoplasm, however, the viruses mature by budding through the plasma membrane. The next two groups, orthomyxoviridae and paramyxoviridae, are surrounded by a membrane. They also contain RNA polymerase and neuriminidase within the virion, have a helical nucleoprotein core, and some replicate in the nucleus. The last family, retroviridae, are the most complex. They contain two or more molecules of RNA, one molecule of reverse transcriptase, many proteins, and membranes. Replication occurs entirely in the nucleus.
TABLE 3
Parvoviridae Adenoviridae
Tobamovirus Picornaviridae Picornaviridae Picornaviridae Picornaviridae Togaviridae Picornaviridae Picomaviridae Togaviridae Covirus Paramyxoviridae Togaviridae Picornaviridae Rhabdoviridae Poxviridae Paramyxoviridae Orthomyxoviridae Orthomyxoviridae Retroviridae Herpetoviridae Retroviridae Retroviridae Papavaviridae Retroviridae Comovirus Retroviridae
Family RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA DNA RNA RNA RNA RNA DNA RNA RNA DNA RNA RNA RNA RNA DNA DNA
Nucleic acid
TDerived from electrophoretic and sucrose gradient S values. $.Oncogenic.
Tobacco mosaic Nodamura Encephalomyocarditis Rhinovirus Mengovirus Sindbis Columbia SK Foot & Mouth Eastern equine encephalitis Bean pod mottle Sendai Semliki forest Polio Vesicular stomatitis Vaccinia Newcastle disease Influenza Fowl Plague Murine sarcomat Herpes simplex3 Murine leukemia sarcoma$ Mouse leukemia2 sv 401 Rous sarcomat Cowpea mosaic Visnal: Mason Pfizer agent$ Adeno associated AdenovirusS
Virus
;i 75-125 100-150 120-130 SO-200 loo-150 100-200 16Ot 1707 170 150-200 170-200 200 200 200 200 150-250
80-t
4: 38-48 SO-55 30-80 30-SO? 60-80 70 55-95’r
0
Poly(A) size
Fraser, 1973 Neuman & Brown, 1976 Frisby, Smith, Jeffers & Porter, 1976 Nair & Panicala, 1976 Marshall & Arlinghaus, 1976 Deborde & Leibowitz, 1976; Eaton & Faulkner, 1972 Johnston & Rose, 1972 Chatterjee, Bachrach & Polatnik, 1976 Armstrong et al., 1972 Semancik, 1974 Pridgen & Kingsbury, 1972 Clegg & Kennedy, 1974 Armstrong et al., 1972; Yogo & Wimmer, 1972 Soria & Huang, 1973; Rose & Knipe, 1975 Nevins & Joklik, 1975; Kates & Beeson, 1970 Weiss & Bratt, 1974 Etkind & Krug, 1974 Ghendon & Blagovesbienskaya, 1975 Green & Cartas, 1972 Bachenheimer & Roizman, 1972 Ross, Tronick & Scholnick, 1972 Lai & Duesberg, 1972 Weinberg, Ben-Ishai & Newbold, 1972 Wang & Duesberg, 1974; Lai & Duesberg, 1972 Manna & Bruening, 1973 Gillespie et al., 1973 Gillespie et al., 1973 Carter, 1976 Philipson et al., 1971
References
Relationship of poly(A) and viruses. Average poly(A) sizesfor difSerent viruses have been listed in increasing order
EVOLUTION
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The parallel increase in poly(A) size and “evolutionary complexity” in eucaryotic viruses suggests two possible modes of evolution of viruses. The fitst possible mode for the evolution of viruses would involve the evolution of viruses parallel with eucaryotic cells. The poly(A) size of the simplest viruses, picornaviridae, have an average poly(A) size of about 50 AMP units which is approximately the poly(A) of the primitive eucaryotes. Since there are smaller poly(A) sizes within the family, the eucaryotic viruses could have evolved from procaryotic cells or procaryotic RNA phages. The evolution of the viruses to more complex forms would then occur as eucaryotic cells evolved to more and more complex forms. The second possible mode of evolution for viruses could be that viruses could have evolved at different times. As eucaryotic cells became more evolved, viruses which formed from the cell machinery (protovirus theory, Temin, 1974) would have poly(A) sizes approximately equal to the cell at that stage of evolution. A problem with this theory is that viruses, after spontaneously forming from a eucaryotic cell must cease to evolve at a significant rate. Another relationship which is apparent in Table 3 is that animal viruses with poly(A) segments of about 200 are generally oncogenic. If poly(A) size does represent an evolutionary pattern in viruses, it would indicate oncogemc viruses are the most recent to develop. One reason these viruses would tend to have poly(A) segments of this length would result from the ability of these viruses to produce mRNA using the cells machinery. On the basis of poly(A) no distinction can be made on whether oncogenic viruses originated according to the protovirus theory of Temin or by continuous chain of evolution. By using the poly(A) size of viruses the following evolutionary pattern emerges (Fig. 2). The Crst organism to have evolved would be a virus-like
I 0
I 50
I 100
I
1
150
200
Poly (A) size
FIG. 2. Proposed evolution of viruses. Arrows indicate the direction of evolution. Viruses and eucaryotic cells are proposed to be evolving in parallel. Interactions between the viruses and cells are assumed to occur at all times. Dotted lines indicate possible pathways. The starred pathway indicates the evolution of RNA phages from nonpolyadenylated bacterial mRNA, not a reverse evolutionary process.
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structure. This is likely since a nucleic acid-protein complex would be easier to form from prebiotic materials than would a completely metabolically active cell. The previrus could “parasitize” the prebiotic ocean in order to replicate and evolve. The previrus would slowly evolve into a procaryotic cell. Later the previrus would evolve into DNA bacteriophages. These bacteriophages would “parasitize” the bacteria as the prebiotic ocean became depleted of organic molecules. RNA bacteriophages, because of their mRNA characteristics, probably evolved from bacterial mRNA. This would also be likely since RNA would be very unstable in the prebiotic ocean. As procaryotic cells developed into eucaryotic cells the resulting gross changes within the cells would prevent the infection of the cells by bacteriophages. Bacteria would then begin to invade eucaryotic cells. Some of these invasions might have resulted in the formation of mitochondria and chloroplasts. In other cases a portion of the bacterial cell machinery could have evolved into primitive eucaryotic viruses. At this stage, the evolution of viruses from procaryotes is more likely than the formation of protoviruses from eucaryotic cells since the replication of simple viruses occur in the cytoplasm. This indicates that the nuclear cell machinery of the developing eucaryotic cell is incompatible with viral replicative mechanisms, as would be the case for a procaryotic replicative mechanism in a eucaryotic cell. An observation from Table 3, which is directly applicable to the evolution of viruses is the difference in poly(A) size between RNA and DNA viruses. DNA viruses (average poly(A) size-171) do not appear until after a poly(A) size of greater than 100 is reached whereas RNA viruses with poly(A) sizes less than 100 are abundant. This would indicate that RNA animal viruses developed first. In the case of the formatiion of the first eucaryotic virus from bacterial cell machinery, it would seem reasonable that RNA would be involved in viral evolution instead of bacterial DNA. Bacterial DNA would have no relationship with the DNA replicating machinery of the cell whereas bacterial mRNA is similar to eucaryotic mRNA. Thus by the addition of a single protein, a RNA directed RNA polymerase, a mechanism for the replication of viral RNA outside the nucleus would be present. Also consistent with the formation of animal viruses from bacterial mRNA is a pathway involving RNA phages. This would be essentially the same pathway except the formation of RNA viruses would occur first. Thus a bacterial mRNA could form a RNA bacteriophage which would continue to evolve and later invade early eucaryotes. The RNA viruses would continue to evolve in a parallel manner as the eucaryotic cells. During evolution, mechanisms probably developed in cells which accelerated evolution. One of these mechanisms might include the use of reverse transcriptase (Temin, 1974). When RNA viruses infected these
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cells, DNA copies suitable for manipulation in the cell nucleus would be generated. Also some DNA viruses could have been formed from these transcripts. Table 2 indicates that DNA viruses and RNA viruses containing reverse transcriptase have approximately the same average poly(A) size (DNA viruses-171, Retroviridae-175) indicating their evolution at approximately the same time. The RNA viruses, after having the DNA copies incorporated into the genome, could detach from the genome with parts of the eucaryotic genome. Eventually the viruses would become integrated with some control elements and thus aquire the ability to alter cells (oncogenic viruses). According to natural selection a virus which could induce a cell to increase its rate of division would be more likely to survive since more copies of itself would be present without destroying the host. If this theory is correct more refined measurements of poly(A) size could be used to determine at what point in evolution reverse transcriptase developed. A comparison of poly(A) and evolution have produced the following general conclusions: (1) poly(A) h as evolved in length ; (2) poly(A) is characteristic of many different classes of organisms ; (3) mitochondrial poly(A) is evolving to longer lengths; (4) poly(A) appears to regulate the stability of mRNA ; and (5) poly(A) size can be used to study the evolutionary order of viruses. As further poly(A) size determinations are made these tables can be refined and thus portray a more precise picture of what has been proposed in this paper. One of the most fruitful areas for poly(A) determinations is in the field of eucaryotic viruses. As more poly(A) sizes are determined a better evolutionary relationship can be drawn between other families and orders of viruses. Also the evolutionary relationships between plant, protist, and animal viruses can be determined more precisely. Since poly(A) sizes tend to remain the same for individual families, i.e. picornaviridae, poly(A) size could be used to help classify viruses. Thus the relationship between poly(A) size and evolution offers a new taxonomic method to study the evolution of viruses. I wish to thank Dr A. P. Kimball for valuable discussions and editorial assistance. Thanks are also due to Dr W. R. Jeffery and Mr D. S. Adams for helpful suggestions. REFERENCES ADAMS, D. S. & JEWERY, W. R. (1977). Fed. Proc. 36,2617. ARMSTRONG, J. A., EDMONDS, M., NAKAZATO, H., PHILLIPS, B. A. & VAUGHAN, (1972). Science 176, 526. AVN, H., VOLOCH, Z., BASTOS, R. & LEVY, S. (1976). Cell 8,495. BACHENHEIMER, S. L. & ROIZMAN, B. (1972). J. Virol. 10, 875. BAGLIONI, C., PEMBERTON, R. & DELQVITCH, T. (1972).FEBS Lett. 26, 320. BERGER, S. L. & COOPER, H. L. (1975).Proc. mtn. Acad. Sci., U.S.A. 72, 3873.
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Note added in proof The log of average poly(A) size for different organisms has been recently shown to have evolved in a linear manner with the time of origin for the organisms (Carlin, in prep.).