Cell, Vol. 5, 51-58,

May

1975,

Copyright%

1975

by MIT

The 5’ Terminal Structure of the Methylated mRNA Synthesized in Vitro by Vesicular Stomatitis Virus Gordon Abraham, Dennis P. Rhodes, and Amiya K. Banerjee Department of Cell Biology Roche Institute of Molecular Biology Nutley, New Jersey 07110

Summary The 5’ terminal structure of the mRNA synthesized in vitro by the virion-associated RNA poiymerase of vesicular stomatitis virus in the presence of Sadenosyi-L-methionine consists of 7-methyl guanosine linked to 2’-O-methyl adenosine through a 5’-5’ pyrophosphate bond as The (Y and p phosphates of GTP m7G(s),,,(5pQ ... and (Y phosphate of ATP are incorporated into the blocked 5’ terminal structure. introduction Vesicular stomatitis virus (VSV) contains a virionassociated RNA polymerase (Baltimore, Huang, and Stampfer, 1970) which in vitro synthesizes multiple mRNA species which contain poly(A); the combined molecular weights of these molecules correspond to the size of the entire RNA genome (Banerjee and Rhodes, 1973; Banerjee, Moyer, and Rhodes, 1974; Moyer and Banerjee, 1975). In addition to the transcriptase activity, a methyl transferase activity was recently detected in purified VSV which incorporates methyl groups derived from Sadenosyl-L-methionine (SAM) into the 5’ termini of the 12-18s VSV RNA products (Rhodes, Moyer, and Banerjee, 1974). Similar enzyme activities have also been demonstrated in cytoplasmic polyhedrosis virus (CPV) (Furuichi, 1974) reovirus (Shatkin, 1974) and vaccinia virus (Wei and Moss, 1974). Although the precise sites of methylation in VSV in vitro mRNA were not known, evidence for the presence of 2 methyl groups per molecule, one of which was a2’-O-methyl ribonucleotide at the 5’ terminus, was reported (Rhodes et al., 1974). In this communication we present a detailed analysis of the structure of the methylated 5’ termini of the 1218s VSV in vitro RNA. The results indicate that the methylation occurs at the 2’-ribose moiety of the 5’ terminal base of the RNA chains with a second methylated nucleotide linked to it through a 5’-5’ pyrophosphate bond. Results Analysis of the 5’ Termini of the Methylateci VW 12-18s RNA Products Methylated VSV mRNA was synthesized in vitro in the presence of the four ribonucleoside triphos-

phates and 3H-SAM. The RNA was extracted with phenol followed by Sephadex G-100 column chromatography, and the 12-18s RNA species were purified by gradient centrifugation. The 3Hlabeled RNA was then digested with RNAase T2 (which degrades RNA to 3’ nucleotides), and the resulting products were analyzed on a DEAEcellulose column by elution with a linear gradient of NaCI. As shown in Figure 1 A, a single peak of 3H radioactivity eluted just after the -5 charged oligonucleotide marker. This labeled material was desalted and a portion treated with bacterial alkaline phosphatase and reanalyzed. A single peak of radioactivity eluted between charges -3 and -4, consistent with the removal of a single phosphate group (Figure 1B). This result indicated that other phosphate groups in the methylated oligonucleotide(s) were resistant to alkaline phosphatase digestion. Another portion of the material eluting at -5 charge was treated with penicillium nuclease [which degrades RNA to 5’ mononucleotides, regardless of the presence of any 2’-O-methyl groups, and also contains a 3’-phosphatase activity (Fujimoto, Kuninaka, and Yoshino, 1974a, b)] and analyzed by DEAE-cellulose chromatography. The elution position of the 3H radioactivity shifted to approximately the -2.5 charge position (Figure lC), consistent with the removal of a phosphate and a 5’ nucleotide from the methylated oligonucleotide(s) (-5 charge). The results indicate that the methylated 5’ terminal structure of VSV mRNA is apparently blocked because it contains phosphate groups which are resistant to phosphatase and phosphodiesterase actions. The 3H-labeled 5’ terminal oligomer (Figure 1A) was further analyzed by high voltage paper electrophoresis. After treatment with penicillium nuclease and alkaline phosphatase, the resultant material migrated on paper near the pA marker (Figure 2A). This material was eluted from the paper and one aliquot treated with nucleotide pyrophosphatase and another with nucleotide pyrophosphatase followed by alkaline phosphatase, and both the samples were analyzed separately by paper electrophoresis. As shown in Figure 2B, treatment with nucleotide pyrophosphatase released two equallylabeled products, one of which stayed at the origin and the other comigrated with pA. On the other hand, treatment with a combination of nucleotide pyrophosphatase and alkaline phosphatase released two 3H-labeled products which comigrated towards the cathode with authentic 7-methyl guanosine and adenosine (or 2’-O-methyl adenosine) markers (Figure 2C). The material at the origin in Figure 2B is presumably 7mGMP, since alkaline phosphatase treatment of this material released a single 3H-labeled product

Cell 52

03%

. -1 ’

j

c;’ 0

’

I 0.3 ‘u 0

I-

_---

_ ----- /_---

_----

__A--

_*-

_---!+-?+r-.-

C

I

Origin

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o-2 = E 0.1 s-

n

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*-

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X 2-

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-i-i

comigrating with m7G (data not shown). By the same criteria, the 3H-labeled compound migrating with pA in Figure 28 seems to be 2’-O-methyl AMP. m7GMP stays at the origin during electrophoresis at pH 3.5 because the negative charge of the phosphate is neutralized by the positive charge on the purine due to methylation at the 7 position. The methylated nucleosides (Figure 2C) were further analyzed by paper chromatography as shown in Figure 3. Methylated adenosine comigrated with the 2’-O-methyl adenosine marker (Figure 3A), and the methylated guanosine comigrated with the 7-methyl guanosine marker (Figure 3B), confirming the identities of these two methylated compounds. It is important to note that at the alkaline pH used in this chromatographic system, m7G (both the marker and the labeled nucleoside) is converted to the ring opened structure: 2-amino-4-hydroxy-

-i

-i

I ?-6-

-1 -1 -i -[

41

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cI

7mG

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1

00

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I 8 I I 40 60 FRACTION NUMBER

Figure 1, DEAE-Cellulose Chromatography Products Following Enzymatic Digestions

I

80

of W-Methylated

,I

100 J

RNA

VSV mRNA was synthesized in vitro using 3H-SAM as the labeled substrate, and the 12-19s mRNA was purified and digested with RNAase T2 as described in Experimental Procedures, After column chromatography on DEAE-cellulose, a portion of each fraction was analyzed for radioactivity (A). The fractions containing radioactivity were desalted by dialysis, and a portion of the RNAase TP-resistant oligonucleotides digested with alkaline phosphatase prior to rechromatography (B). Another portion of the RNAase TP-resistant oligonucleotides was digested with penicillium nuclease prior to rechromatography (C). The salt concentrations at which radioactive peaks eluted were 0.13, 0.09, and 0.07 M NaCl for (A), (B), and(C), respectively. Arrows indicate the elution positions of marker oligonucleotides expressed as their net negative charges.

0 Figure 2. Paper of IH-Methylated

I

IO 20 FRACTION NUMBER Electrophoretic RNA Products

Analyses

of Enzymatic

30 Digestions

A sample of the SH-labeled oligonucleotides eluting from DEAEcellulose at -5 charge (Figure 1 A) was digested with penicillium nuclease followed by alkaline phosphatase, prior to analysis (A). After electrophoresis, the radioactivity was eluted from the paper and one portion digested with nucleotide pyrophosphatase (B), and another digested with nucleotide pyrophosphatase followed by alkaline phosphatase (C). prior to reanalysis. Marker nucleotides and nucleosides were included in each sample, and their positions after electrophoresis are shown.

5’ Terminal 53

Structure

of Methylated

In Vitro

mRNA

5-(N-methyl) carboxamide-6-ribosylamino pyrimidine. The identity of the m7G was further confirmed using a different chromatographic system at acid pH (data not shown). The results clearly demonstrate that the 5’ termini consist of two methylated bases linked with a pyrophosphate bond. The tentative structure of the penicillium nuclease and phosphatase-resistant material can be depicted as m7GcpppjAF or Amcpppjm7G. The corresponding structures of the -5 charged material after RNAase T2 digestion (compare with Figure 1 A) would be m7GrpppjA$Xp and Am(pppj7mGpXp, which would elute at -5 charge because of the positive charge carried by m7G. Conflrmation of Phosphatase Resistance of the 5’ Terminal Phosphates of the RNA Product. The phosphatase resistance of the 5’ terminal phosphates of the RNA products can be directly demonstrated using ,l3, +*P-labeled nucleoside triphosphates as labeled substrates. We have previously shown that /3, y-3*P GTP was incorporated into the I

IA

-

I

?mG

I A

I 2L0-mA

5’ termini of methylated mRNA products (Rhodes et al., 1974). RNA labeled with p, y-3*P GTP and 3H-methyl SAM was purified by phenol extraction and gradient centrifugation. The double-labeled RNA was digested by RNAase T2 and analyzed by DEAE-cellulose chromatography. All of the radioactivity eluted as oligonucleotides at -5 charge (compare with Figure 1A). This material was then treated with various enzymes and the products were analyzed by paper electrophoresis as shown in Figure 4. The untreated material (eluting at -5 charge) migrated on paper near pG (Figure 4A). Treatment with penicillium nuclease and alkaline phosphatase did not release any 3*P as P, (Figure 48) and both the 3H and 3*P radioactivities comigrated near pA as observed earlier (compare with Figure 2A). However, 80% of the 3*P radioactivity was released as Pi after treatment of the original material with nucleotide pyrophosphatase alone (Figure 4C). The nature of the 3*P radioactivity remaining at the origin is unclear. The 3H radioactivity, on the other hand, appeared as two products, one remaining at the origin presumably as m7GMP, and the other migrating near pU, presumably as ,AFX~. Thus the re-

1

i

0

:: 0

IO FRACTION

Figure 3. Nucleosides

Paper

Chromatographic

20

30

NUMBER Analyses

of

‘H-Methylated

‘H-labeled nucleosides from Figure 2C were eluted from the paper and analyzed by descending paper chromatography using isopropanol:ammonia:water (7:1:2) as the solvent. Authentic samples of P’-0-methyladenosine and 7-methyl guanosine were included with the samples and located by ultraviolet absorption. The positions of other marker nucleosides are shown. (A) Chromatography (6) Chromatography

of the methylated of the methylated

adenosine guanosine

derivative. derivative.

-----7

0

----

------

I

--_a-

IO FRACTION

L-

_---1

----

---------

20 NUMBER

Figure 4. Paper Electrophoretic Analyses of Enzymatic 3H-Methylated RNA Also Labeled with p,y-“P GTP

,

0

30 Digests

of

VSV mRNA was synthesized in vitro using ‘H-SAM and B,y-QP GTP as the labeled substrates, and purified as described in Experimental Procedures. Oligonucleotides resistant to RNAase T2 and labeled with both 3H and 32P were isolated from a DEAE-cellulose column, charge (compare with Figure 1A). These eluting at -5 oligonucleotides were either analyzed directly by electrophoresis (A), digested with penicillium nuclease followed by alkaline phosphatase (B), or digested with nucleotide pyrophosphatase (C) prior to analysis.

Cell 54

sults confirm that the 5’ terminal phosphates are resistant to alkaline phosphatase treatment and are present in a pyrophosphate linked structure. Confirmation of Methylated Bases Using a-3zP-Labeled ATP and GTP In order to demonstrate that the methylated nucleosides are indeed derived from guanosine and adenosine, methylated RNAs were synthesized in vitro using either (Y-~*P GTP or a-32P ATP as labeled substrates in separate reaction mixtures containing 3HSAM. The RNA products were purified as described earlier and the oligonucleotides released upon RNAase T2 digestion were purified by DEAEcellulose chromatography. Using either ,-3*P GTP or a-3*P ATP, most of the 3*P radioactivity eluted as mononucleotides at -2 charge. In each case an additional peak of radioactivity eluted at -5 charge (compare with Figure 1A) containing both 3H and 3*P (data not shown). The material eluting at -5

charge using 3H-SAM and (Y-~*P GTP as labeled substrates was further analyzed as shown in Figure 5. After treatment with penicillium nuclease and alkaline phosphatase, the 3H and 3*P radioactivities migrated together near the pA marker (Figure 5A) as noted earlier (compare with Figure 48). This material was eluted from the paper and treated with nucleotide pyrophosphatase. One portion was analyzed directly (Figure 58) and the other digested with alkaline phosphatase prior to electrophoresis (Figure 5C). As observed earlier, after treatment with nucleotide pyrophosphatase alone, 3H radioactivity was associated with two principal products, one staying at the origin as m7GMP and the other migrating with pA as 2’-0-mAMP. The 3*P radioactivity, on the other hand, was recovered mainly with m7GMP. A small portion of each isotope comigrated near pG (Figure 5B) and was believed to be m7GDP, resulting from incomplete digestion by nucleotide pyrophosphatase. This material was subsequently

60

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r

I

1

:I Origin

3

F

I

n-l

3

Figure 5. Paper of ‘H-Methylated

PI

j /

- I2

I5 IO

FRACTION

I

rl

NUMBER

Electrophoretic Analyses of Enzymatic RNA Also Labeled with a-‘zP GTP

Digestions

VSV mRNA was synthesized in vitro using ‘H-SAM and a-3zP GTP as the labeled substrates, and purified as described in Experimental Procedures. Oligonucleotides resistant to RNAase T2 and labeled with both 3H and 3zP were isolated from a DEAE-cellulose column, eluting at -5 charge (compare with Figure 1 A). These oligonucleotides were digested with penicillium nuclease followed by alkaline phosphatase prior to analysis (A). After electrophoresis, the radioactivity was eluted from the paper and one portion digested with nucleotide pyrophosphatase(B). and another digested with nucleotide pyrophosphatase followed by alkaline phosphatase (C), prior to reanalysis.

FRACTION Figure 6. Paper of 3H-Methylated Experimental ure 5 except

NUMBER

Electrophoretic Analyses of Enzymatic RNA Also Labeled with a-32P ATP

Procedures that n-‘ZP

Digestions

were identical to those described in FigATP was substituted for a-“P GTP.

(A) Oligonucleotides resistant to penicillium nuclease followed by alkaline phosphatase. (B) The material from (A) digested with nucleotide pyrophosphatase. (C) The material from (A) digested with nucleotide pyrophosphatase followed by alkaline phosphatase.

5’ Terminal 55

Structure

of Methylated

In Vitro

mRNA

shown to be wholly sensitive to phosphatase, releasing m7G and Pi (data not shown). When the material in Figure 5A was treated with both nucleotide pyrophosphatase and alkaline phosphatase, all of the QP radioactivity appeared as Pi and the 3H radioactivity comigrated with m7G and 2’-O-mA (Figure 5C). These results, together with those shown in Figure 4, demonstrate that one of the methylated bases was derived from GTP with both the (Y and ,L?phosphates incorporated into the blocked 5’ terminal structure. The same experiments were done with RNA products labeled with JH-SAM and (Y-QP ATP, and the results are shown in Figure 6. In contrast to the 3H-SAM and (~-32P GTP labeled RNA, when the n-32P ATP-labeled material eluting at -5 charge was treated with penicillium nuclease and alkaline phosphatase, 50% of the 32P radioactivity was released as Pi, and the entire 3H and the other 50% of the 32P radioactivity migrated together at the characteristic position near pA (Figure 6A). The release of the 50% of the 3*P radioactivity at Pi indicated that either the second or the third base from the 5’ terminus is adenosine (see below). As shown in Figure 6B, when the material resistant to penicillium nuclease and alkaline phosphatase was treated with nucleotide pyrophosphatase, all of the 3*P radioactivity and 50% of the 3H radioactivity was converted to a product comigrating with pA, presumably as 2’-0-mAMP. The other 50% of 3H radioactivity remained at the origin, presumably at m7GMP. On the other hand, the same material when treated with both nucleotide pyrophosphatase and alkaline phosphatase released the entire QP radioactivity as Pi, while the 3H radioactivity comigrated with 2’-OmA and m7G (Figure 6C). From the results shown in Figures 4, 5, and 6, the most likely structure of the 5’ termini of the methylated RNA is m7G&,aAmX . . . , where there are a minimum of three pho:phPates resistant to penicillium nuclease and alkaline phosphatase. The pyrophosphate linkage is derived from the (Y and p phosphates of GTP and the N phosphate of ATP. The fact that p,y-32P ATP was not incorporated into the RNA products by VSV RNA polymerase (data not shown) is consistent with this structure. Also, since 50% of the 32P radioactivity in the -5 charged material was released as Pi in Figure 6B, one of the other two phosphates in the proposed structure is derived from the (Y phosphate of ATP. 5’4’ Linkage Between the Terminal Nucleotides The observation that the (Y both GTP and phosphate of ATP were incorporated into the 5’ terminal structure suggested that an unusual 5’-5’ linkage existed between the two bases, that is, m7G~S,,pptis,A$Xp. . If this is the case, the blocking base (m7G) will con-

tain free 2’-and 3’-hydroxyl groups, and be subject to periodate oxidation and removal by treatment with aniline. This will result in the removal of m7G, and the P-eliminated RNA will contain 5’-,.,,,A,mX, ., which upon treatment with penicillium nuclease will be converted into pppAm. To test this prediction, RNA synthesized in the presence of 3H-SAM was purified and oxidized with potassium periodate, followed by treatment with aniline. The remaining RNA product was digested with penicillium nuclease and the digest analyzed on a DEAE-cellulose column. As shown in Figure 7, 30% of the 3H radioactivity eluted between charges -2 and -3, while 70% eluted after the ADP marker and almost identically with authentic CI-~*P ATP marker. When the latter material, including a-32P ATP, was desalted and treated with alkaline phosphatase, the 32P radioactivity was totally converted into Pi, and the 3H radioactivity into 2’-O-mA (Figure 8). No m7G was detected. From these results we concluded that the blocking group was m7G containing free 2’- and 3’-hydroxyl groups, since periodate oxidation and /3 elimination converted the 5’ terminal structure into pppA~Xp. . . . The 3H radioactivity eluting between charges -2 and -3 presumably resulted from incomplete p elimination (compare with Figure 1 C). The results showed that there were three phosphates present in the 5’ terminal structure which were linked with methylated guanosine and adenosine through a 5-5’ linkage. The presence of such a linkage was further shown by treatment of the m7GMP and the 2’-0-mAMP, released by pyrophosphatase digestion of the penicillium nuclease and phosphatase resistant compound (compare with Figure 2B), with 5’ or 3’ nucleotidases. Both the methylated nucleotides were dephosphorylated only by the 5’ nucleotides (data not shown), confirming that the linkage between guanosine and adenosine was 5’-5’. Discussion The 12-18s VSV mRNA, synthesized in vitro in the presence of the methyl donor SAM by the virionassociated RNA polymerase of VSV, contained an unusual 5’ terminal structure: m7Grr,~I,,,(s~,Apm. . The structure was deduced on the basis of the following observations: -the 3*P from p,y-32P GTP incorporated into the methylated 5’ termini was resistant to alkaline phosphatase, indicating that the 5’ termini were blocked; -this phosphatase-resistant phosphate group was released as Pi by treatment with nucleotide pyrophosphatase alone, indicating that a pyrophosphate linkage existed in the blocked structure; -the methylated nucleosides released from the blocked structure were identified as m7G and 2’-0-

Cell 56

mA by paper electrophoresis and chromatography; -the removal of m7G by periodate oxidation and p elimination indicated that it was the blocking base and also that it contained free 2’- and 3’-hydroxyl groups. It was further shown to be linked to the 5’ position of 2’-O-mA by a pyrophosphate bridge containing three phosphate groups. In the present studies, this structure was shown to be present at the 5’ termini of the 12-18s in vitro VSV-methylated mRNAs, which comprise four of the five VSV mRNA species (Both, Moyer, and Banerjee, 1975). The 5’ terminal structure of the methylated 31s in vitro mRNA (Rhodes et al., 1974; Moyer and Banerjee, 1975) is currently under investigation. 5’ terminal structures similar to that found in VSV mRNAs have recently been shown to be present in mRNAs made in vitro by other viruses which contain virion-associated polymerases. These include reovirus, m7Grs)ppp(5,,G3 (Furuichi et al., 1975a), cytoplasmic polyhedrosis virus, m7G15,)pppr5,1A~. (Furuichi and Miura, 1975), and vaccinia virus, m7G,5~~prp~p~~~~G~. . and m7G,5~~,~,~,r5~~A,m.. . (Urushi-

bara et al., 1975; Wei and Moss, 1975). Methylated mRNAs have also been demonstrated in mouse L cells (Perry and Kelley, 1974) and Novikoff hepatoma cells (Desrosiers, Friderici, and Rottmann, 1974). Recently, several low molecular weight RNAs isolated from Novikoff hepatoma cell nuclei have been shown to contain 5’ terminal m+2~7G,5~,pp(s,IAp (Reddy et al., 1974). Methylated and blocked 5’ termini have also been found in viral specific mRNAs in vivo as shown for VSV (Moyer et al., 1975) and SV40 (Lavi and Shatkin, personal communication). CPV and reovirus genome RNAs have been shown to contain blocked 5’ termini in one of the strands (Miura, Watanabe, and Sugiura, 1974; Furuichi, Muthukrishnan, and Shatkin, 1975b). In addition, the blocked 5’ termini of reovirus genome RNA are also methylated (Furuichi et al., 1975b). The in vitro synthesis of the blocked 5’ terminal structure by VSV appears to involve several identifiable processes including: methylation of a specific guanosine at the 7 position; methylation of a specific adenosine at the 2’ position; the formation of a specific 5’-5’ pyrophosphate linkage [the “cap-

7

PPA 7

0

1

20

40

60

80‘

FRACTION NUMBER Figure

7. DEAE-Cellulose

Chromatography

of ‘H-Methylated

RNA

after /J’ Elimination

and Penicillium

Nuclease

Digestion

VSV mRNA was synthesized in vitro using XH-SAM as the labeled substrate, and purified as described above. The RNA was oxidized with potassium periodate followed by treatment with aniline as described in Experimental Procedures. After ethanol precipitation, the modified RNA was digested with penicillium nuclease for 3 hr and mixed with a sample of authentic a-32P ATP prior to chromatography. A portion of each fraction was analyzed for the presence of both isotopes. (0 --- 0) ‘H radioactivity; (0 --- 0) 32P radioactivity.

5’ Terminal 57

Structure

of Methylated

In Vitro

mRNA

ping” reaction (Rottman, Shatkin, and Perry, 1974)]; and formation of phosphodiester bonds during transcription, that is, the polymerization reaction. The order of these reactions, the number of separate enzymes involved, and the reaction mechanisms have not been determined. In this regard, it is interesting to note that when VSV cores, isolated by polyethylene glycol-dextran phase separation, were used to synthesize RNA in vitro (in the absence of SAM), the RNA products contained 5’ terminal pppAp. . . and pppGp. . (Roy and Bishop, 1973). This suggests that the “capping” reaction may be catalyzed by a separate enzyme and is not required for polymerization. Similarly, the methylation activity that is located mostly in the dextran phase with the cores (Rhodes et al., 1974) cannot be required for polymerization since RNA can be easily made in the absence of SAM. Recently we have observed that RNA made by triton-disrupted VSV in the absence of SAM is also blocked at the 5’ termini (Abraham, Rhodes, and Banerjee, 1975), indicating that methylation is not a prerequisite for the “capping” reaction. Further experiments aimed at the separation and purification of these various enzyme activities and the mechanism of the reactions involved are currently being pursued. The significance of this kind of 5’ terminal structure in eucaryotic and viral mRNAs is still a matter of speculation (Rottman et al., 1974). Since a wide

A 40

I

I Origin

FRACTION

I 8

NUMBER

Figure 8. Paper Electrophoretic Analyses of the Presumptive rriphosphate Obtained after Penicillium Nuclease Digestion of the P-Eliminated RNA The major peak of radioactivity from the previous figure (fractions 37-43, including the marker a-3zP ATP) was desalted and (A) electrophoresed directly or(B) treated with alkaline phosphatase prior to electrophoresis.

variety of cellular and viral mRNAs possess this special structure, it seems likely that it confers a specialized form or function to the mRNAs. As a possible role of methylation, it has been shown recently that methylation of reovirus and VSV mRNAs appears to be required for the initiation of their translation in vitro (Both, Banerjee, and Shatkin, 1975). Future studies will shed light on the possible functions of methylated and blocked 5’ termini of mRNAs in eucaryotic organisms. Experimental

Procedures

Purllicatlon of VSV VSV (Indiana serotype) was grown in baby hamster (BHK 21, clone 13, adapted to suspension culture) as described previously (Banerjee et al.. 1974).

kidney cells and purified

Synthesls and Purlflcation of RNA In Vitro RNA was synthesized in vitro in a standard incubation mixture as described previously (Rhodes et al., 1974), except that when labeling with LY-~ZP GTP or /3,y-‘2P GTP, the concentration of GTP was 0.1 mM. Incubation was at 30°C for 2 hr. The reaction was terminated by the addition of sodium dodecyl sulfate to 0.5%. and the product RNA was directly extracted with phenol and purified by Sephadex G-100 chromatography and by SDS-sucrose gradient centrifugation as detailed previously (Banerjee and Rhodes, 1973; Rhodes et al., 1974; Moyer and Banerjee, 1975). The RNA species sedimenting between 12 and 16s were pooled and precipitated with ethanol. Enzyme Treatment of RNA Products RNAase T2 Labeled RNA was dissolved in 0.5 ml 10 mM sodium acetate (pH 4.5) together with 50 rg of carrier yeast tRNA and 2.5 units of RNAase T2. Digestion was for 5 hr at 37°C. Baclerial Alkaline Phosphalase The incubation mixture contained 20 mM Tris-HCI (pH 6.0) and 20 units/ml of bacterial alkaline phosphatase. Digestion was for 30 min at 37°C. Penicillium Nuclease The incubation mixture contained 10 mM sodium acetate (pH 6.0) and 200 pg/ml of penicillium nuclease. Digestion was for 30 min at 37°C. Nucleotide Pyrophosphatase The incubation mixture contained 20 mM Tris-HCI (pH 7.5), 1 mM MgC12, and 0.05 units/ml nucleotide pyrophosphatase. Digestion was for 15 min at 37°C. DEAE-Cellulose Chromatography and Hlgh Voltage Paper Electrophoresls Procedures used for DEAE-cellulose chromatography have been described previously (Banerjee and Shatkin, 1971). Appropriate fractions were pooled and desalted by exhaustive dialysis at 4°C against water and lyophilized (Banerjee and Shatkin, 1971). High voltage paper electrophoresis on Whatman No. 3 MM paper was at 2600V for 40 min in pyridine-acetate buffer (pH 3.5) (Banerjee, Rensing, and August, 1969). Appropriate marker compounds were included with the samples and located by ultraviolet light. The paper was cut into 1 cm strips and radioactivity determined by scintillation counting. Periodate Oxldatlon and Removal of the Blocking Base The procedure for periodate oxidation and p elimination tially as described by Hunt (1970). Labeled RNA samples solved in 0.5 ml 0.1 M sodium acetate buffer (pH 5.3) 1 mM EDTA. Freshly prepared potassium periodate

by Anlllne was essenwere discontaining solution in

Cell 58

water was added to a final concentration of 0.4 mM and incubated at room temperature in the dark for 2 hr. Excess periodate was decomposed by adding glucose to 1 mM and the RNA precipitated with ethanol. After centrifugation. the pellet was dissolved in 0.5 ml 0.33 M aniline (prepared by diluting 30 fold with water and adjusting the pH to 5.0 with 2 N HCI) and incubated in the dark for 2 hr. The RNA was precipitated with ethanol, and the pellet was washed twice more with ethanol. The RNA, with its blocking group removed by aniline, was digested with penicillium nuclease and analyzed by DEAE-cellulose chromatography. Chemicals and Enzymes S-adenosyl-L-PH-methyl methionine (8.3 Ci/mmole), a-3*P GTP (20.4 Ci/mmole), and rx-32P ATP (23.1 Ci/mmole) were purchased from New England Nuclear, Boston, Mass. p,y-QP GTP (4.7 Ci/ mmole) was purchased from International Chemical and Nuclear, Irvine, Calif. Bacterial alkaline phosphatase (BAPF) was purchased from Worthington Biochemical Corp., Freehold, N.J., RNAase T2 from Sankyo Co., Japan, and nucleotide pyrophosphatase from Sigma Chemical Co., St. Louis, MO. Penicillium nuclease was kindly provided by Dr. Kuninaka, Yamasa Co., Ltd., Japan. Methylated nucleoside markers were obtained from P-L Biochemical.% Inc., Milwaukee, Wis. and Sigma Chemical Co., St. Louis, MO. Acknowledgment We wish to thank Drs. S. Moyer, A. J. Shatkin, Y. Furuichi, and S. Muthukrishnan for their help and many stimulating discussions. Received

January

28, 1975;

revised

February

21, 1975

References Abraham, in press.

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D. P., and Banerjee,

A. K. (1975).

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D. P. (1973).

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A. J. (1971). U., and

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Rottman,

F. (1974).

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Fujimoto. M., Kuninaka, Chem. 38, 785.

A., and

Yoshino,

H. (1974a).

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Biol.

Fujimoto. M., Kuninaka, Chem. 38, 1555.

A., and

Yoshino,

H. (1974b).

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Furuichi.

Y. (1974).

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Y., and Miura.

Nucleic

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Res.

Furuichi, (1975a).

Y., Morgan, M., Muthukrishnan. S., and Proc. Nat. Acad. Sci. USA 72, 362.

K.-l. (1975).

7, 609. Nature

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