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5'-Terminal Capping of RNA by Guanylyltransferase

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5'-Terminal Capping of RNA by Guanylyltransferase Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3758-3761, September 1977 Biochemistry 5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei (mRNA/7-methylguanosine/RNA processing) CHA-MER WEI AND BERNARD MOSS Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Robert P. Perry, June 24, 1977 ABSTRACT A soluble extract prepared from HeLa cell MATERIALS AND METHODS nuclei has been shown to catalyze the 5'-terminal modification Preparation of Guanylyltransferase from of RNA and synthetic polyribonucleotides to form m7G(5')pppA- HeLa Cells. and m7G(5')pppG- structures referred to as caps. The reaction HeLa S3 cells were harvested from suspension culture at 3 X involves the transfer of a GMP moiety from GTP to the 5' end 105 cells per ml and washed three times with 20mM Tris-HCl of an RNA molecule containing at Teast two terminal phos- (pH 7.6)/146 mM NaCl/11 mM glucose at 4°. The cells were phates. Significantly, neither the P nor the 'y phosphates of GTP then swollen in three volumes of 20mM Tris-HC1 (pH 7.9)/10 are transferred and polynucleotides with no 5'-terminal phos- mM MgCI2/10 mM KCI/0.25 mM spermidine at 00 and dis- phate or only one are not acceptors. In the absence of methyl rupted by Dounce homogenization. An equal volume of cold donor, G(5')pppA- and G(5')pppG- structures were synthesized, indicating that methylation is not required for guanylylation. 20mM Tris.HCI (pH 7.6)/50% glycerol was immediately added Cap formation was considered to occur by the following to the lysate. The nuclei were pelleted by centrifugation, re- mechanism: suspended in buffer A [50 mM Tris-HCl (pH 7.9)/25% glyc- 'yfla a ,fy erol/i mM dithiothreitol/0.1 mM EDTA], and layered over pppG + (p)ppN- -_ G(5')pppN- + PPi [1] 50 mM Tris-HCI (pH 7.9)/10 mM MgCl2/50% glycerol. The nuclei were pelleted again by centrifugation at 37,400 X g for G(5')pppN- + AdoMet -_ m7G(5')pppN- + AdoHcy [2] 30 min, resuspended in buffer A, and lysed by adding 3 M in which AdoMet is S-adenosylmethionine, AdoHcy is S- (NH4)2S04 (adjusted to pH 7.9) to a final concentration of 0.3 adenosylhomocysteine; and (p)ppN- represents either the orig- M. The nuclear lysate was dispersed by six 10-sec bursts with inal 5' end of an RNA molecule or an internal site to which one a Branson sonifier at 00, and the supernatant obtained by cen- or more phosphates were added after processing. trifugation at 234,000 X g for 50 min was used as the enzyme source. Structures of the type m7G(5')pppNmp(Nm)-, known as caps, Guanylyltransferase Assay. The standard reaction mixture are present at the 5' termini of viral (1-4) and cellular (5-9) (0.1 ml) containing 50 mM Tris-HCl (pH 7.9), 5 mM MgCl2, mRNAs. A comprehensive review of the occurrence and 0.5 mM MnCl2, 1 mM ATP, 1 mM dithiothreitol, 12.5% glyc- function in translation of cap structures has recently been erol, 10 ,g of RNA substrate, 20 ,ul of enzyme extract, 25-50 published (10). Information regarding the mechanism of for- MCi of [a-32P]GTP (100-200 Ci/mmol) and 100,uM S-adeno- mation of these structures has been obtained only for those vi- sylmethionine (AdoMet) (50MM added at 0 min and 50MM at ruses that contain the capping and methylating enzymes within 30 min) or 200 ,M S-adenosylhomocysteine (AdoHcy) was the purified virus particles. Two general mechanisms have been incubated at 300 for 1 hr. The reaction was stopped by addition found. With vaccinia virus (11-13), cytoplasmic polyhedrosis of 1 ml of 50 mM Tris-HCI (pH 7.9), 0.5% sodium dodecyl virus (14), and reovirus (15), a GMP residue from GTP is sulfate, 0.1 mM GTP, 1 mM EDTA, and 0.3 mg of yeast tRNA transferred to the 5' terminus of mRNA, while with vesicular carrier. The mixture was extracted with chloroform/buffer- stomatitis virus (16), a GDP residue is transferred. The first saturated phenol (1:1), and the labeled RNA was isolated by mechanism is suitable for capping the initial triphosphate end precipitation of the aqueous phase with 5% trichloroacetic acid. of an RNA molecule, while the second seems appropriate for The precipitate was redissolved in minimal Tris-HCI (pH 8.5) capping monophosphate ends formed by cleavage. and the precipitation procedure was repeated three times. The Elucidation of the mechanism of capping cellular mRNA final precipitate was washed with 95% ethanol. Alternatively, species is important for understanding RNA processing. The the RNA, after chloroform/phenol extraction, was separated presence of caps in heterogeneous nuclear RNA (17, 18) implies from the majority of unreacted [a-32P]GTP by gel filtration. that this modification occurs within the nucleus. Moreover, The labeled RNA was digested in 0.1 ml of 10mM sodium ac- subcellular fractions containing nuclei have been shown to etate (pH 6) with 50,ug of nuclease P1 at 370 for 2 hr and then synthesize capped mRNA in vitro (19, 20). The requirement in 50 mM Tris-HCl (pH 8.5)/5 mM MgCl2 with 20 Mg of for the presence of all four ribonucleoside triphosphates and Escherichia coli alkaline phosphatase at 370 for 2 hr. The digests the sensitivity to a-amanitin suggested that cap formation is were analyzed by paper electrophoresis. closely coupled to transcription in this system (20). The ex- Electrophoresis and Chromatography. Paper electropho- periments described here, which utilize soluble enzymes ob- resis was performed on 57-cm sheets of Whatman 3 MM paper tained from HeLa cell nuclei and defined RNA substrates, in 0.5% pyridine/5% acetic acid (pH 3.5)/1 mM EDTA buffer. provide evidence regarding a mechanism of capping cellular Ascending paper chromatography was in isobutyric acid/0.5 mRNA. M NH40H (5:3). The costs of publication of this article were defrayed in part by the Abbreviations: m7G, 7-methylguanosine; Am, 2'-O-methyladenosine; payment of page charges. This article must therefore be hereby marked Gm, 2'-0-methylguanosine; m6Am, N6,2'-O-dimethyladenosine; Nm, "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate any 2'-0-methylnucleoside; AdoMet, S-adenosylmethionine; AdoHcy, this fact. S-adenosylhomocysteine. 3758 Downloaded by guest on September 28, 2021 Biochemistry: Wei and Moss Proc. Natl. Acad. Sci. USA 74 (1977) 3759 A plus AdoHcy 8 GpppG GpppA 0 8OL~ ' x E a C.) B mlGpppGm plus AdoMet 15 mTGpppAm 0M m'GpppG \ m7GpppA mlGpppAm x 4 - 6..~ 0 0 -o 10 20 30 m7GpppGm c Fraction number 515 mGpppAmn Piii FIG. 2. Identification by paper chromatography of methylated and unmethylated caps. The experiment was carried out as in Fig. 1 10 except that labeled RNA was separated from unreacted [a-32P]GTP by gel filtration instead of acid precipitation. (A) The material mi- 5 -origin Pi grating with and slightly ahead of the marker, as in Fig. 1C, was eluted and chromatographed in isobutyric acid/NH40H. (B) The material migrating with the marker, as in Fig. 1B, was eluted and chromato- 0 10 20 30 40 Fraction number i graphed. FIG. 1. Paper electrophoresis of nuclease P1- and phosphatase- resistant products of guanylyltransferase reaction. Guanylyltrans- [a-32PJGTP into nuclease P1- and alkaline phosphatase-resistant ferase was measured with [a-32P]GTP and unmethylated vaccinia material when RNA was omitted from the reaction (Fig. IA). mRNA in the presence ofAdoMet or AdoHcy. After phenol extraction In the presence of AdoHcy, a methylation inhibitor, [a-32P]GTP and trichloroacetic acid precipitation, the RNA was digested with was incorporated predominantly into material migrating nuclease P1 and alkaline phosphatase and the products were analyzed slightly ahead of the methylated cap markers (Fig. 1C). The by paper electrophoresis at pH 3.5. Methyl-3H-labeled m7GpppGm difference in migration is consistent with the absence of the and used as internal markers were derived from m7GpppAm at 7 methyl-3H-labeled vaccinia mRNA synthesized by virus cores. (A) positive charge associated with methylation the position Minus vaccinia RNA plus AdoMet; (B) plus vaccinia RNA and of guanine. AdoMet; (C) plus vaccinia RNA and AdoHcy. The Products of Guanylyltransferase. Nuclease PI- and alkaline phosphatase-resistant oligonucleotides labeled with Materials. Vaccinia mRNA was synthesized as described by [a-32P]GTP were isolated by paper electrophoresis as described Ensinger et al. (11). [a-32P]GTP (150 Ci/mmol) was purchased in the preceding section. For further characterization, the la- from either New England Nuclear Co. or Amersham/Searle beled oligonucleotides were chromatographed in an isobutyric Corp.; [ribose-3H]GTP (23 Ci/mmol) from Amersham/Searle acid/NH40H system. As shown in Fig. 2A, the material syn- Corp.; [/,By-32P]GTP (24 Ci/mmol) from International thesized in the presence of AdoHcy cochromatographed with Chemical and Nuclear Corp.; poly(A) lot no. 341101 and G(5')pppG and G(5')pppA. However, the material synthesized methylated nucleotide markers from P-L Biochemicals; E. coli in the presence of AdoMet cochromatographed with m7G- alkaline phosphatase from Worthington Biochemical Corp.; (5')pppG and m7G(5')pppA (Fig. 2B). Since the unmethylated nuclease P1 from Yamasa Shoyu Co.; and nucleotide pyro- vaccinia virus mRNA contains both ppA and ppG ends, the phosphatase from Sigma Chemical Corp. A second phosphate above results were consistent with the transfer of the GMP was added to the 5' terminus of poly(A), as described by Martin moiety of GTP to the ends of the substrate to form the appro- and Moss (12).
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