Cell, Vol . 9, 6 4 5 -653, December 1976 (Part 2), Copyright ©1976 by MIT

Review

Capping of Eucaryotic mRNAs

A . J . Shatkin Roche Institute of Molecular Biology Nutley, New Jersey 07110

Messenger RNAs (mRNAs) are synthesized, translated, and possibly also degraded in a 5'-3' direction (Perry, 1976 ; Lodish, 1976) . Consequently, their 5' terminal structures are of some interest, with possible implications for regulation of genetic expression . In procaryotes, the 5' end of many mRNAs is a triphosphorylated purine corresponding to the residue that initiated transcription . Even in cases in which procaryotic messages are processed by cleavage from a larger precursor, the processed message retains a 5' terminal phosphate . By contrast, most eucaryotic cellular and viral mRNAs recently have been found to be modified at the 5' end . The modification consists of a "cap," m 7 G(5')ppp(5')X, that protects the mRNA at its terminus against attack by phosphatases and other nucleases, and promotes mRNA function at the level of initiation of translation (Rottman, Shatkin, and Perry, 1974 ; Shatkin, 1976) . Cap Structure The general structural features of the 5' cap of eucaryotic mRNAs are shown in Figure 1 . The terminal m 7 G and the penultimate nucleotide are joined by their 5'-hydroxyl groups through a triphosphate bridge . This 5'-5' linkage is inverted, relative to the normal 3'-5' phosphodiester bonds in the remainder of the polynucleotide chain . As a consequence of the inversion of the terminal m 7 G residue, the capped end of the message has a 2',3'-cis,diol that provides the basis for several chemical manipulations of caps . For example, since free 2',3'-hydroxyls can form complexes with borate ions, caps or capped oligonucleotides can be purified by affinity chromatography on substrates such as dihydroxyboryl-cellulose (Furuichi et al ., 1975c) . The cis,diol is oxidized by periodate treatment to the dialdehyde which can then be reduced with 3Hborohydride to label the terminus (Furuichi, Muthukrishnan, and Shatkin, 1975a ; Muthukrishnan et al ., 1975a) . Alternatively, exposure of the oxidized mRNA to aniline causes /3 elimination of the m 7 G dialdehyde, yielding a 5'-triphosphate-ended RNA molecule (Furuichi et al ., 1975b ; Wei and Moss, 1975) . A common property of caps is the presence of 7-methylguanosine . N 7 -methylation of guanosine results in the acquisition of a positive charge that partially neutralizes the phosphate groups in m 7 GpppX, which therefore has a net negative charge of between -2 and -3 . In contrast to N7 methylated deoxyguanosine, the glycosidic bond of

the ribonucleoside is not labilized by methylation . However, the 8-hydrogen becomes exchangeable, and mRNA caps synthesized with 8- 3 H-GTP as precursor are not labeled in the 5' terminal m 7 G . In addition, under alkaline conditions, the imidazole ring is opened at the 8-9 bond, converting m 7 G to the ring-opened derivative, 2-amino-4-hydroxy-5(N-methyl)carboxamide-6-ribosylamino-pyrimidine, which lacks the positive charge . While capped messages terminate in m 7 G, the nature of the penultimate residue varies, both in the identity of the base and in the occurrence of methylation in this position . Certain mRNAs from animal (Dubin and Stollar, 1975 ; Colonno and Stone, 1975, 1976 ; Hefti et al ., 1976) and plant viruses (Pinck, 1975 ; Zimmern, 1975 ; Symons, 1975 ; Keith and Fraenkel-Conrat, 1975a ; Dasgupta, Harada, and Kaesberg, 1976), as well as cellular mRNAs from yeast (Sripati, Groner, and Warner, 1976 ; Mager, Klootwijk, and Klein, (1976), and the majority of slime mold messages (Dottin, Weiner, and Lodish, 1976) contain m 7 GpppX-that is, a "cap zero" that is not methylated further . Other mRNAs (Furuichi et al ., 1975b ; Wei and Moss, 1975 ; Adams and Cory, 1975) have an additional methyl group on the 2' position of the penultimate residue to form m 7 GpppXm, the "cap 1" structure . In such caps, the 3'-5' bond between the 2'-O-methylated penultimate nucleotide and the next residue in the mRNA chain is stabilized against hydrolysis by alkali or T2 RNAase because formation of the 2',3' cyclic intermediate required for cleavage is blocked . Thus while T2 RNAase digestion releases m 7GpppXp from cap zero-terminated messages, the structure m 7 GpppXm-Yp is released from cap 1-terminated messages . In type 1 capped structures that have Am in the position of X, base methylation at the 6 position can also occur . The resulting cap, m 7 Gpppm 6 Am, is present in a fraction of the mRNAs of several different mammalian cells (Adams and

BASE H2 N

0 0 0 5 II II II 5 CHz 0-P-0-P-0-P-0-CH 2 0 I I I 0- 000(CH3) BASE, 0 I 5' O=P-0-CH? 0 0-

Figure 1 . Structure of 5' Cap

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Cory, 1975 ; Moyer and Banerjee, 1976 ; Dubin and Taylor, 1975 ; Wei, Gershowitz, and Moss, 1975b) and animal viruses (Moyer and Banerjee, 1976 ; Wei et al ., 1975b ; Sommer et al ., 1976 ; Moss and Koczot, 1976 ; Krug, Morgan, and Shatkin, 1976) . 2'-0methylation of the third residue from the 5' end of many mRNAs, including silkworm fibroin (Yang, Manning, and Gage, 1976) and mammalian cell mRNA (Cory and Adams, 1975), results in a still more extensively modified "cap 2 ." RNAase or alkaline digestion of mRNA with a cap 2 structure yields a 5' terminal tetranucleotide m 7 GpppXm-Ym-Zp . In addition to the methylated nucleotides at the 5' terminus, internal m 6 A (ti1/1500 nucleotides) and smaller amounts of m 5 C (Dubin and Taylor, 1975 ; Sommer et al ., 1976) are present in some eucaryotic cellular (Adams and Cory, 1975 ; Desrosiers, Friderici, and Rottman, 1974 ; Lavi and Shatkin, 1975) and viral (Furuichi et al ., 1975c ; Sommer et al ., 1976 ; Moss and Koczot, 1976 ; Krug et al ., 1976 ; Lavi and Shatkin, 1975) mRNAs . Distribution Blocked and methylated 5' termini, almost identical to mRNA caps except for the presence of 5'M3 2,2,7G, were first observed in low molecular weight RNAs of rat hepatoma nuclei (Shibata et al ., 1975) .

With the exception of these low molecular weight nuclear RNAs, caps are a peculiarity of messenger RNA and its presumptive precursor, heterogeneous nuclear RNA . Capping does not occur on ribosomal or transfer RNAs . Furthermore, among RNA viruses in which the genome RNA has minus polarity, it is not the RNA present in the virion which is capped . Rather, caps are present in the messenger RNAs which are complementary to the genome of these viruses . In the case of adenovirus-infected cells, adenovirus messenger RNAs are capped, but a low molecular weight, nonmessenger RNA (VA-RNA) is uncapped (McGuire, Piatak, and Hodge, 1976) . Caps have been detected in almost all eucaryotic viral mRNAs, as indicated in Table 1 . Exceptions include picornavirus (Nomoto, Lee, and Wimmer, 1976 ; Hewlett, Rose, and Baltimore, 1976 ; Fernandez-Munoz and Darnell, 1976 ; Nuss et al ., 1975) and satellite tobacco necrosis virus (STNV ; Wimmer et al ., 1968) RNAs, which apparently contain a 5' terminal mono- or disphosphate . In the case of polio virus, the uncapped ends of the RNA may be blocked in some other way, for example, by a viral protein (E . Wimmer, personal communication) . All capped viral mRNAs contain exclusively a purine in the 5' penultimate position . The extent of methylation of this penultimate residue varies . For exam-

Table 1 . Eucaryotic Viral mRNAs with a 5' Terminal Cap, m'GpppX Type

Reference

DNA Vaccinia

Wei and Moss, 1975 ; Urushibara et al ., 1975

SV40

Lavi and Shatkin, 1975 ; Aloni, 1975

Adeno

Sommer et al., 1976 ; Moss and Koczot, 1976 ; Wold, Green, and Munns, 1976

Herpes

B . Clements, I . Hay, and A . Shatkin, unpublished results

RNA Single-Stranded Arbo-Sindbis

Dubin and Stollar, 1975 ; Hefti et al ., 1976

Arbo-Semliki Forest

P . Fellner, personal communication

Rhabdo-Vesicular Stomatitis

Abraham et al ., 1975a ; Rose, 1975 ; Moyer and Banerjee, 1976

Paramyxo-Newcastle Disease

Colonno and Stone, 1975, 1976

Myxo-Influenza

Krug et al ., 1976

Avian Oncorna-Rous

Furuichi et al ., 1975c; Keith and Fraenkel-Conrat, 1975b ; Stoltzfus and Dimock, 1976

Murine Oncorna-Moloney

Bondurant, Hashimoto, and Green, 1976 ; Rose, Haseltine, and Baltimore, 1976

Plant-Tobacco Mosaic

Zimmern, 1975 ; Keith and Fraenkel-Conrat, 1975a

Plant-Brome Grass Mosaic

Dasgupta et al ., 1976

Plant-Alfalfa Mosaic

Pinck, 1975 ; Roman et al ., 1976

Plant-Cucumber Mosaic

Symons, 1975

RNA Double-Stranded Human-Reo

Furuichi et al ., 1975b ; Faust et al., 1975a

Insect-Cytoplasmic Polyhedrosis

Furuichi and Miura, 1975

Plant-Rice Dwarf

Miura et al ., 1975

Plant-Wound Tumor

D . P . Rhodes, D . V . R . Reddy, L . M . Black, and A . K . Banerjee, submitted for publication

Capping of Eucaryotic mRNAs 6 47

pie, type zero caps (m 7 GpppG) are found in tobacco mosaic virus (TMV) and Brome mosaic virus (BMV ; Dasgupta et al ., 1976 ; Zimmern, 1975 ; Keith and Kraenkel-Conrat, 1975a) and in Sindbis virus RNA (m 7 GpppA ; Hefti et al ., 1976 ; Dubin and Stollar, 1975) . Monomethylated type 1 caps are present in in vitro transcripts of reovirus (m 7 GpppGm ; Furuichi et al ., 1975b ; Faust, Hastings, and Millward, 1975a), vesicular stomatitis virus (VSV, m 7 GpppAm ; Abraham, Rhodes, and Banerjee, 1975a), and vaccinia virus (m 7 GpppAm and m 7 GpppGm ; Wei and Moss, 1975) . In contrast to the simple cap 1 structures found on these RNAs transcribed in vitro by virionassociated enzymes, messenger RNAs extracted from virus-infected cells include both type 1 and type 2 caps in the case of VSV (Moyer and Banerjee, 1976 ; Rose, 1975), vaccinia (B . Moss, personal communication), and reovirus (Y . Furuichi and A . Shatkin, unpublished results) . A mixture of cap structures (m 7 GpppAm, m 7 Gpppm 6Am, m 7 GpppAmNm ; Sommer et al ., 1976 ; Moss and Koczot, 1976) has also been found on adenovirus messenger RNAs extracted from infected cells . Among eucaryotic cells, all mRNAs analyzed appear to be capped . A wide range of mRNA sources (Table 2) have been tested, including cells of vertebrate and invertebrate origin . In mammalian cell mRNAs, the penultimate residue X in m 7 GpppX can be a pyrimidine as well as purine . By contrast, mRNAs of those lower forms that have been examined [silkworm (Yang et'al ., 1976) ; sea urchin (Surrey and Nemer, 1976 ; Faust, Millward, and Fromson, 1975b ; G . Giudice, personal com-

munication) ; brine shrimp (Muthukrishnan et al ., 1975b ; Y . Groner, personal communication) ; slime mold (Dottin et al ., 1976) ; and yeast (Sripati et al ., 1976 ; Mager et al ., 1976) ; De Kloet and Andrean, 1976)] have only purines at the 5' end . This observation may imply that synthesis of purine-containing caps occurs during the initiation of transcription, while a cleavage-related mechanism is used for generating pyrimidine-containing caps . In addition, it is interesting to note that the complexity of the cap increases as one moves up the evolutionary scale from yeast (cap zero) through slime mold (75% cap zero, 20% cap 1), brine shrimp larvae and sea urchin embryos (exclusively cap 1), silkworm (fibroin, cap 2), and mammals (high percentage of cap 1 and cap 2) . The relative amounts of cap 1 and cap 2 observed in mammalian cells varied in different studies, and in yeast mRNA, type zero (Sripati et al ., 1976 ; Mager et al ., 1976) and type 1 caps (De Kloet and Andrean, 1976) were reported by different investigators . Some of these variations may be a function of the labeling conditions used (Perry and Kelley, 1976 ; Friderici, Dovenberg, and Rottman, 1976) . Mechanism of Synthesis The reaction series involved in the synthesis of caps has been worked out most completely for several viral mRNAs . These studies have been facilitated because purified virions of many animal viruses contain all the enzyme activities required for the formation of capped mRNA . Incubation of disrupted vaccinia virus (Wei and Moss, 1975 ; Urushibara et

Table 2 . Eucaryotic Cellular mRNAs with a 5' Terminal Cap, m 7 GpppX Type

Reference

Human-HeLa Line

Furuichi et al ., 1975d ; Wei, Gershowitz, and Moss, 1975a, 1976

Monkey-BSC-1 Line

Lavi and Shatkin, 1975

Rabbit-Reticulocyte

Muthukrishnan et al ., 1975a ; Hunt and Oakes, 1976

Hamster-BHK Line

Dubin and Taylor, 1975 ; Moyer and Banerjee, 1976

Rat-Hepatoma

Desrosiers, Friderici, and Rottman, 1975

Mouse-L Line

Wei et al ., 1975b ; Perry et al ., 1975a, 1975b

Mouse-Myeloma

Adams and Cory, 1975 ; Cory and Adams, 1975

Mouse-Erythroid

W . L . Heckle, R . G . Fenton, T . G . Wood, C . G . Merkel, and J . B . Lingrel, submitted for publication

Mouse-Kidney

Ouellette, Frederick, and Malt, 1975

Duck-Erythroid

Perry and Scherrer, 1975

Chicken-Ovalbumin

H . Busch, personal communication

Toad

M . Crippa, personal communication

Silkworm-Fibroin

Yang et al ., 1976

Brine Shrimp

Muthukrishnan et al ., 1975b

Sea Urchin

Faust et al ., 1975b; Surrey and Nemer, 1976

Slime Mold

Dottin et al ., 1976

Yeast

Sripati et al ., 1976 ; Mager et al ., 1976 ; De Kloet and Andrean, 1976



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al ., 1975), VSV (Abraham et al ., 1975a), or chymotrypsin-digested reovirus (Furuichi et al ., 1975b ; Faust et al ., 1975a) under appropriate conditions in a defined reaction mixture containing ribonucleoside triphosphates and the methyl donor, S-adenosylmethionine (SAM), results in the synthesis of capped mRNA . In reovirus mRNA, cap synthesis apparently occurs at the initiation of transcription after the first phosphodiester bond has been formed between GTP and CTP by the virion-associated RNA polymerase (Figure 2 ; Furuichi et al ., 1976) . The resulting pppG-C is converted to ppG-C by a virion nucleotide phosphohydrolase and then capped by guanosine monophosphate transfer from GTP, with accompanying release of inorganic pyrophosphate . This blocking reaction is inhibited by high levels of pyrophosphate, and the back reaction, GpppG-C ppG-C + GTP, is correspondingly promoted by pyrophosphate (Furuichi and Shatkin, 1976) . Thus reovirus mRNA synthesized in the presence of 2 mM pyrophosphate contains mainly 5' terminal ppG-C, while mRNA made with inorganic pyrophosphatase in the reaction mixture has 5'-GpppG-C . When the incubation mixture also contains SAM, this structure is methylated at the N7 position of the 5' terminal G, a modification that renders the m 7 GpppG-C resistant to pyrophosphorolysis . 2'-O-methylation of the penultimate G follows after the formation of m 7 GpppG-C, probably concomitantly with chain growth . No further methylations occur in vitro, but reovirus mRNA isolated from infected mouse L cells contains the cap 2 structure, m 7 GpppGm-Cm-U, in approximately half the molecules . Presumably, the 2'-O-methylation of C is catalyzed by a cellular enzyme . A similar reaction series, involving transfer of pG from GTP to ribopolymers containing 5'-ppG or ppA, has been established for vaccinia virus cap synthesis (Ensinger et al ., 1975 ; Moss et al ., 1976) . The enzymes that synthesize vaccinia caps have been solubilized and purified (Martin, Paoletti, and Moss, 1975 ; Martin and Moss, 1975) . The guanylyl transferase and N 7 -methylase probably exist as a (I)

Phosphodiester bond formation pppG + pppC ) pppG-C + PPi RNA polymerase

(2)

Nucleotide phosphohydrolase reaction pppG-C -) ppG-C + Pi

(3)

Capping PPG-C + pp*pG

(4)

7

` GpppG-C + PPi EguanyIyl transferase

N -methylation GpppG-C + SAM ---N m GpppG-C + SAH (cap zero)

7

(5) 2'-0-methylation m CpppG-C . . . + SAM -N m GpppGWC + SAH (cap I) -----------(6) Second 2'-0-methylation (not catalyzed by virion-associated enzyme) nSGpppGOC . . . + SAM ) GpppG°Cm . . . + SAH (cap 2) cellular 2'-0-methylase

7

7

m7

Figure 2 . Mechanism of Reovirus mRNA Cap Synthesis

complex of two polypeptides of molecular weights 97,000 and 31,400 daltons . A separate enzyme mediates 2'-O-methylation . Vaccinia and reovirus are both capable of capping exogenous polymers and thus could act post-transcriptionally . However, reovirus mRNA is normally capped at the level of the nascent dinucleotide . It seems possible that vaccinia mRNA may also be capped at the initiation of transcription . Cytoplasmic polyhedrosis virus mRNA is also capped at the start of mRNA synthesis (Shimotohno and Miura, 1976), and transcription in vitro by purified virions is dependent upon the presence of SAM (Furuichi, 1974) . Cellular mRNAs that are derived from the initiated 5' end of a transcript may be modified by a mechanism similar to that employed by reovirus and vaccinia . However, a different mechanism would be required for those cellular mRNAs that contain m 7 GpppPyrimidine, since these are presumably derived, by cleavage, from internal regions of a larger precursor molecule . Precedence for capping of a 5' monophosphate end, such as would be expected from cleavage, may come from the studies on VSV mRNA synthesis (Abraham, Rhodes, and Banerjee, 1975b ; Colonno and Banerjee, 1976 ; Abraham and Banerjee, 1976 ; Ball and White, 1976) . Recent findings suggest that the formation of VSV mRNA may occur by a cleavage scheme similar to that observed for T7 bacteriophage early mRNA (Dunn and Studier, 1973), but with the embellishments of capping, methylation, and 3' polyadenylation . The origin of the phosphates in the VSV cap, m 7 GPP I PA, and other results suggest that its synthesis may proceed as follows : cleavage pppA-N-N I pA-N . . . - ppA-N-N + pA-N . . . (1) pA-N . . . +'p ppG GpppA-N . . . + Pi (2) GpppA-N . . . + SAM-m 7 GpppAm-N . . . + SAH (3) It is presently unclear whether either or both the initiation-related and proposed cleavage-dependent mechanisms for viral mRNA cap synthesis are employed for generation of cellular mRNAs, but evidence has been obtained recently that both mechanisms may operate in mouse L cells (Schibler and Perry, 1976) . Capped, high molecular weight, heterogeneous RNAs of both the 3'-polyadenylated and nonadenylated classes are present in nuclei of mammalian tissue culture cells (Perry and Kelley, 1976 ; Salditt-Georgieff et al ., 1976 ; Perry et al ., 1975b), and isolated HeLa cell nuclei synthesize caps in vitro (Groner and Hurwitz, 1975) . The smaller nuclear RNAs (ti25S) isolated from HeLa cells include a higher proportion of capped molecules than the fast sedimenting (>40S) RNAs, and nuclear RNAs similar in size (10S-20S) to cytoplas-

Capping of Eucaryotic mRNAs 64 9

mic mRNA have an average of about one cap per RNA chain, as does messenger RNA (Salditt-Georgieff et al ., 1976) . Kinetic studies of 3 H-methyllabeled L cell RNA indicate that caps on nuclear transcripts are conserved and appear in cytoplasmic RNA (Perry and Kelley, 1976) . This result indicates that the 5' ends of some nuclear RNAs and mRNAs are the same . Only type 1 caps are present in nuclei . In some molecules, they are converted to cap 2 structures in the cytoplasm by an additional 2'-O-methylation (Perry and Kelley, 1976 ; Friderici et al ., 1976) . Consistent with the conversion of nuclear transcripts to cytoplasmic mRNAs, radioactive 5' triphosphate termini were detected in nuclear but not cytoplasmic poly(A)+ RNAs from mammalian cells pulse-labeled with 32 P (Georgiev et al ., 1972 ; Schmincke, Herrmann, and Hausen, 1976) . The shortest labeling times resulted in nuclear RNA with the highest proportion of uncapped ends, and this proportion decreased with longer labeling times . A striking similarity was observed in the composition of the caps of polyadenylated nuclear heterogeneous RNA and cytoplasmic mRNA of mouse L cells consistent with a precursor-product relationship between the two in mammalian cells (Perry et al ., 1975b) . The similarity in size and cap composition of nuclear transcripts and cytoplasmic mRNA in slime mold (Dottin et al ., 1976) suggests that in this organism, the conversion is a direct one involving few or no cleavages . The precise mechanisms required for generating capped, functional cellular mRNAs remain to be established . Function Consistent with their ubiquity in eucaryotic mRNA, caps have an important facilitating role in translation . This effect on function was first demonstrated with reovirus and vesicular stomatitis virus mRNAs which required 5' terminal m7G in caps for efficient translation in a wheat germ cell-free, proteinsynthesizing system (Both, Banerjee, and Shatkin, 1975a) . In an in vivo situation-that is, VSV-infected BHK cells-the importance of caps for translation was suggested by the observation that all polysome-associated viral RNA was capped, while a small proportion of the viral RNA that was not directing viral protein formation contained uncapped triphosphate 5' ends (Rose, 1975) . A similar selection by ribosomes of capped over uncapped messenger molecules has been demonstrated in vitro by incubating a mixture of pppGm . . . and m 7 GpppGm . . .-ended reovirus RNA molecules in wheat germ extracts (Muthukrishnan et al ., 1976) . As further evidence that the 5' terminal m7G is the functionally important residue in caps, its removal from reovirus mRNA, rabbit reticulocyte mRNA (Muthukrishnan et al ., 1975a), and in more recent

studies, silk fibroin mRNA (P . Gage, personal communication) and bovine parathyroid mRNA (Kemper, 1976) was accompanied by a marked decrease in ability to direct protein synthesis in wheat germ extract . Similar findings were obtained with cellular and viral mRNAs in cell-free translating systems prepared from brine shrimp (A . salina) (Muthukrishnan et al ., 1975b) and rabbit reticulocytes (Rose and Lodish, 1976), indicating that normally, capped mRNAs containing 5' terminal ppX, pppX, pppXm, or GpppX are translated considerably less efficiently in vitro than mRNAs with m 7 GpppX or m 7 GpppXm . However, the dependence of eucaryotic mRNA translation on cap appears to be neither absolute nor universal . Poliovirus RNA isolated from polyribosomes of productively infected HeLa cells contained 5' terminal pU (Nomoto et al ., 1976 ; Hewlett et al ., 1976 ; Fernandez-Munoz and Darnell, 1976) . RNAs from purified poliovirus or another picornavirus, EMC, also do not contain 5' linked terminal m 7 G, but are translated efficiently in mammalian cell extracts (Lodish, 1976 ; Nuss et al ., 1975) . Unmethylated early mRNAs of T3 and T7 bacteriophages can be translated in extracts of mammalian cells or wheat germ, although less efficiently than capped reovirus mRNA (Anderson, Atkins, and Dunn, 1976) . Satellite tobacco necrosis virus RNA has 5' terminal ppA (Wimmer et al ., 1968) and codes for viral coat protein in infected cells and in wheat germ extract ; /3 elimination or addition of S-adenosylhomocysteine (SAH) or SAM to the extract did not significantly alter its translation in vitro (Kemper, 1976 ; Roman et al ., 1976) . Since it has been possible to convert the ppA to m 7 GpppA by incubating STNV RNA with the guanylyl transferase and N 7 Gmethylase activities purified from vaccinia virus (B . Moss, personal communication), it will be of interest to study the effect of this modification on mRNA function . Another plant virus RNA, Brome mosaic virus RNA-4, retained the ability to code for authentic viral coat protein in wheat germ extract after removal of the cap by /3 elimination (Shih, Dasgupta, and Kaesberg, 1976) . Although there was only a 2 fold reduction in translation efficiency when the /3 eliminated RNA was tested at saturating levels, at low RNA concentrations there was an 8 fold reduction in amino acid incorporation after /3 elimination . In the case of VSV mRNA, /3 elimination decreased its translation by 4 fold in reticulocyte lysate and by 10 fold in wheat germ extract (Rose and Lodish, 1976) . The products programmed by the 5'-triphosphate-containing VSV mRNA corresponded to virus-specific structural polypeptides, indicating again that some uncapped mRNAs can be correctly translated in eucaryotic cell-free extracts but with a reduced efficiency .

Cell 650

In reticulocyte lysates, the dependence on 5' terminal m 7 G for stable initiation complex by reovirus and VSV mRNAs was less stringent than in wheat germ extract (Muthukrishnan et al ., 1976) . $- Eliminated, 3 H-methylated mRNAs bound to reticulocyte ribosomes to the extent of about 17% and 34% for reovirus and VSV mRNA, respectively, as compared to values of 70-80% of the input radioactivity in mock-treated, control mRNAs . In another study, the binding to reticulocyte ribosomes of VSV mRNA was reduced from about 50% to 35% after removal of the m 7 G by $ elimination (Rose and Lodish, 1976) . Some, but not all, of the decrease in binding was apparently due to the oxidation step of the R elimination reaction (Muthukrishnan et al ., 1976 ; Rose and Lodish, 1976 ; Rao et-al ., 1975), suggesting that the dialdehyde form of m 7 G in caps may be less readily recognized for ribosome binding or that periodate treatment has nonspecific deleterious effects on mRNA function . The latter seems improbable because periodate oxidation of STNV RNA did not inhibit its translation in wheat germ extract (Kemper, 1976) ; furthermore, when a mixture of oxidized but incompletely eliminated reovirus mRNA was incubated in wheat germ extract, those molecules that retained the oxidized m7G were selectively bound to ribosomes (Muthukrishnan et al ., 1976) . 7-methylguanosine apparently functions at an early stage in the initiation of translation of reovirus mRNA and presumably other viral and cellular mRNAs . Discrimination between methylated and unmethylated reovirus mRNA in wheat germ extract occurs at or before initiation complex formation with 40S ribosomal subunits (Both et al ., 1975b) . In a mixture of molecules with m 7 GpppX and unmethylated and/or unblocked 5' termini of the type, ppG, pppGm, or GpppG, only the m7G-capped mRNA bound efficiently in stable complexes with wheat germ 40S subunits . The selection of molecules with 5'-m 7 G may be mediated by ribosomal proteins and/or soluble protein initiation factors . A . salina extracts contain a ribosome-associated protein(s) that binds m 7 GpppG-C (Filipowicz et al ., 1976) . The binding was strongly competed by m 7 Gcapped mRNAs and by m 7 G-containing cap analogs, but not by EMC, STNV, or 18S ribosomal RNAs, indicating the specificity of the protein for caps . The cap-binding protein did not appear to correspond to purified eucaryotic initiation factors IF-M1, M2A, M2B, M3, M4, M5, or MP . However, in a different type of assay-that is, inhibition of factor binding to capped mRNA by cap analogs-it was reported that the m 7 G in caps is specifically recognized by factor IF-M3 (Shafritz et al ., 1976) . Since this factor also binds to and is required for transla-

tion of uncapped picornavirus RNAs, it presumably is capable of multiple interactions with mRNA . A different type of approach has also provided strong support for the idea that m7G has a functional role in eucaryotic protein synthesis at the level of initiation . Translation of rabbit globin mRNA, tobacco mosaic virus RNA, and HeLa cell poly(A)+ RNA in wheat germ extract was strongly inhibited by m 7 G-5'-monophosphate (mpG) (Hickey, Weber, and Baglioni, 1976a, 1976b) . Since reovirus mRNA binding to 80S ribosomes was >70% inhibited by 0 .5 mM mpG, while translation of STNV RNA and poly(U) was not reduced, the inhibitory effect is highly specific . Similarly, mpG inhibited the mRNAdependent conversion of 40S-Met-tRNA, initiation complexes to 80S complexes in a fractionated wheat germ system when capped mRNAs (TMV, alfalfa mosaic virus RNAs) were used (Roman et al ., 1976) . Inhibition was not obtained with the uncapped STNV RNA . In each case, inhibition was observed with the 5'-monophosphate of m 7 G ; the 3'-phosphate and nucleoside were noninhibitory . In another study (Canaani, Revel, and Groner, 1976), mpG, mppp 5 'G, and m 7 GpppGm, but not m 7 G or GpppGm, markedly inhibited the translation of globin and SV40 mRNA in wheat germ and L cell extracts . Translation in wheat germ extract of bacteriophage T4 mRNA, EMC RNA, and SV40 unmethylated cRNA synthesized in vitro was less inhibited by the cap analogs . Again the effect was demonstrated to be at the level of ribosome binding by testing globin mRNA binding in wheat germ extract . Other positively charged cap analogues, including 7-benzylguanosine diphosphate, inhibited reovirus mRNA binding to wheat germ ribosomes, emphasizing the importance of the extra plus charge in the cap for mRNA function (S . Muthukrishnan, S . Hecht, and A . Shatkin, unpublished results) . Results of nuclear magnetic resonance analysis indicate that an electrostatic interaction between the phosphates and the plus charge of caps leads to a rigid structure that may be preferred for initiation of mRNA translation (E . D . Hickey, L . A . Weber, C . Baglioni, C . H . Kim, and R . H . Sarma, unpublished results) . The m 7 GpppGm terminus of reovirus mRNA comprises part of the binding site for wheat germ ribosome, since the attachment of wheat germ 40S ribosomal subunits to each of the three size classes of reovirus mRNA conferred almost complete protection of the caps against RNAase digestion (Kozak and Shatkin, 1976) . The caps were retained in a unique set of 5' terminal oligonucleotide fragments of chain length ranging from about 31-65 nucleotides from the different mRNAs . The protected fragments recovered from 80S initiation com-

Capping of Eucaryotic mRNAs 651

plexes with reovirus mRNA were found to overlap the 40S-protected sequences, but in some cases, the overlap was incomplete and caps were not retained in the 80S-protected fragments . The protected fragments that retained a cap rebound to form stable 80S ribosome complexes more efficiently (>50%) than those not retaining a cap . However, limited rebinding (11-20%) of uncapped fragments was consistently observed . In addition, 5' terminal, capped oligonucleotides of chain length ti7-10 nucleotides derived from reovirus mRNA by digestion with T1 RNAase did not form stable complexes with wheat germ ribosomes (Both et al ., 1975b) . Thus the presence of cap does not ensure ribosome binding of any oligoribonucleotide, and its absence does not preclude binding . In addition to m 7 G, stable binding to ribosomes apparently also depends upon other mRNA structural features . To study the effects of base composition and 5' terminal structure on mRNA binding to ribosomes, various synthetic ribopolymers were prepared with polynucleotide phosphorylase (Both et al ., 1976) . By using conditions that promoted primer-dependent synthesis, various types of 5' terminal structures were incorporated into polymers of different base composition . Certain ribopolymers, in particular those rich in A and U, bound to wheat germ 80S ribosomes to some extent (11-20%), irrespective of the nature of their 5' ends . Addition of a cap, m 7 GpppGm-C, promoted both the rate and extent (58%) of binding of poly(A •U ) to 80S ribosomes, while addition of 5' terminal GpppG-C, ppG-C, or ring-opened m7G cap did not increase binding . Other capped polymers [poly(C), (A • G), (C • G)] did not form stable 40S or 80S initiation complexes . A third group of capped polymers [poly(U), (A . C), and (U • C)] formed 40S, but only low levels of 80S complexes . Although binding of the uncapped polymers was unaffected by the cap analog, mpG, the extent of binding of capped poly (A2 • U2 . G) was reduced by 1 mM mpG to the level observed for the same polymer containing 5' terminal ppG-C . The results are consistent with the hypothesis that multiple structural features of mRNA, including the cap, internal sequence(s), and possibly the position of the AUG codon, influence stable initiation complex formation . If internal sequences are important for ribosome recognition, there is yet no evidence that the same, or even a similar sequence, will occur in all eucaryotic messages . The finding that the presence of 5' terminal m 7 G promotes stable initiation complex formation and translation of most eucaryotic mRNAs provides a possible site for regulation of genetic expression at the level of translation . Cleavage of mRNA caps would be expected to reduce protein synthesis, a

form of negative control . However, caps apparently are conserved in mammalian tissue culture cell transcripts and degraded in concert with the remainder of the mRNA chain (Perry and Kelley, 1976 ; Ouellette, Reed, and Malt, 1976) . In a cell-free, wheat germ protein-synthesizing system directed by reovirus mRNA, no evidence was obtained for cap turnover during translation (G . Both, unpublished results) . However, cap-hydrolyzing enzymes have been detected in extracts of HeLa (Nuss et al ., 1975) and tobacco cells (Shinshi et al ., 1976) . The HeLa cell pyrophosphatase activity, which cleaves m 7 GpppX to m 7 pG and ppX, has a strict substrate specificity for the positively charged 5' terminal m 7 G . The enzyme has a decreased activity against longer oligonucleotides (chain length ti7-10 nucleotides) and does not attack caps in mRNA . Presumably, the function of the decapping enzyme may be to eliminate residual caps from degraded mRNA fragments and prevent them from inhibiting translation . The tobacco enzyme differs from that of the mammalian cell line . It hydrolyzes various phosphodiester and pyrophosphate bonds including caps, but does not cleave polynucleotides . Thus treatment of cytoplasmic polyhedrosis virus mRNA with the enzyme released mpG from the cap without degrading the RNA chain (Shinshi et al ., 1976) . Recently, it was found that enzymatic removal of the 5' terminal structure from tobacco mosaic virus RNA almost completely destroyed its infectivity (Ohno et al ., 1976) . This enzyme should be particularly useful for studying the effect of "decapping" on mRNA function . Regulation at the level of translation could also be positively controlled at a certain period of development by the occurrence of capping or by methylaation of preexisting capped messages . Unfertilized oocytes of sea urchins and amphibians contain stored maternal mRNA that is utilized for translation within minutes after fertilization . However, most of these "masked" mRNAs appeared to be already capped and methylated since their translation in wheat germ extract was inhibited by mpG and unaffected by addition of SAH or SAM (Hickey et al ., 1976a, 1976b ; Darnbrough and Ford, 1976 ; G . Giudice, personal communication) . Similar results were obtained with mRNAs from A . salina cysts and developing embryos (Muthukrishnan et al ., 1975b) . The stored maternal mRNA of oocytes from an insect, the tobacco hornworm, apparently differs and contains blocked but unmethylated 5' terminal GpppX (Kastern and Berry, 1976) . It will be of interest to study methylation and translation of the hornworm oocyte mRNA . Future studies should help to elucidate the role of the m7G cap in the synthesis, translation, and degradation of eucaryotic mRNA .

Cell 652

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