Methylated, blocked 5' termini of yeast mRNA.

mRNAs of the yeast, Saccharomyces cerevisiae, have modified 5' termini similar but not identical with those present in the higher eukaryotes. Poly(A)-containing RNA isolated from spheroplasts or from polysomes of cells labeled with [methyl-3H]methoionine or 32Pi contain methylated, blocked 5'-terminal structures of composition: m7G(5')pppAp and m7G(5')pppGp, occurring in a relative distribution of 75% and 25%, respectively. Furthermore there is no N6-methyladenosine nor any other methylated nucleoside in the entire molecule of poly(A)-containing RNA. Experiments with a mutant, temperature-sensitive for the transport of mRNA to cytoplasm, suggest that the methylation and addition of the blocking group take place in the nucleus and that the genetic lesion in the mutant does not affect these mechanisms.

). RNA samples were dissolved in 2 to 3 ml of Tris/EDTA/Li/ SDS, LiCl was added to a final concentration of 0.5 M, and the samples were applied to a column (0.5 x 6 cm) of oligo(dT)-cellulose equilibrated-with the binding buffer (Tris/EDTA/L$SDS containing 6.5 M LiCl). The column was washed with 15 ml of binding buffer followed bv 25 ml of binding buffer without SDS. Poly(A)-containing RNA was eluted with 0.5.ml fractions of 10 rnM Tris, pH 7.4. Fractions with poly(A)-containing RNA were pooled, 0.1 volume of 1 M sodium acetate (pH 5.0) was added and the RNA was precipitated with ethanol. Fractions containing RNA that did not bind to oligo(dT)-cellulose were pooled and RNA was precipitated with ethanol. Treatment of RNA with Enzymes-For combined digestion with Penicillium nuclease (P, nuclease, Yamasa Shovu Co.) and bacterial alkaline phosphatase (Worthington Biochemical Corp.), RNA was dissolved in 100 to 150 ~1 of 10 mM sodium acetate buffer (pH 6.0) and incubated with P, nuclkase (1 mg/ml) for 60 min at 37". The pH of the mixture was then brought to 8.0 with 1 M Tris base and incubation was continued for 60 min with bacterial alkaline phosphatase. One unit of enzyme was added four times at 15-min intervals. For RNase T, (Calbiochem) digestion, RNA was dissolved in 100 ~1 of 0.05 M sodium acetate buffer (pH 4.5) and incubated 15 hours at 37" with 5 units of the nuclease. Digestion with nucleotide pyrophosphatase (Sigma Chemical Co.) was carried out for 60 min at 37" in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MgCl, and 0.3 unit of the enzyme/ml.
Chromatography and Paper Electrophoresis-DEAE-cellulose chromatography in 7 M urea was carried out on column (0.7 x 22 cm) equilibrated with 50 mM Tris-HCl (pH 7.6) containing 7 M urea and 5 rnM NaCl. Samples (0.5 to 1 ml) were applied together with tRNA hvdrolysates and eluted with a linear gradient (100 ml) of NaCl (0.005 to 0.25 M in 50 mM Tris-HCl buffer '(pH 7.6) containing 7 M urea). Absorbance at 260 nm was monitored and the radioactive content of each fraction was measured in Aquasol (New England Nuclear, Boston, Mass.).

Paper electrophoresis on Whatman
No. 3MM was carried out at 206 V/cm2 for 2% hours in pyridine-acetate buffer (pH 3.5). The dried paper was cut into l-cm strips, and radioactivity was determined in toluene-based scintillation mixture.

RESULTS
Nuclease-and Phosphatase-resistant 5'-Terminal Structures in 3H-labeled Methylated Poly(A)-containing RNA of Yeast Spheroplasts-Radioactivity is present in poly(A)-containing RNA isolated from spheroplasts exposed to [methyLaH]methionine for 10 min. (This strain is auxotrophic for purines and thus cannot incorporate methyl groups into the purine ring.) This [3H]methyl-labeled RNA was digested with nuclease P, from Penicillium citrinum (which cleaves phosphodiester linkages in polynucleotides, including those containing 2'-0-methylated residues, to yield 5'-nucleotides) (23) followed by bacterial alkaline phosphatase, and the product was analyzed by high voltage paper electrophoresis. As shown in Fig. la, about one-third of the radioactivity in the digest migrated towards the anode as two distinct peaks (I and II) intermediate between the AMP and GMP markers. The remaining radioactivity migrated towards the cathode in spots corresponding to all four ribonucleoside markers. From the specificity of P, and bacterial alkaline phosphatase, peaks I and II must be dinucleotides connected through a pyrophosphate linkage. By analogy with the 5'-terminal structures in mRNA of viruses and mammalian cells, peaks I and II probably arise from the 5' end of poly(A)-terminated RNA molecules. In order to identify their methylated nucleosides, peaks I and II were eluted, digested with nucleotide pyrophosphatase to liberate the mononucleotides, and again analyzed by paper electrophoresis.
The radioactivity from peak I migrated as a spot corresponding to m7GMP except for a trace of m7G (Fig. lb). The radioactivity from peak II also migrated as m'GMP with a trace of m'G (Fig. lc).
The small amount of m'G observed is likely to be due to the dephosphorylation of m'GMP by traces of phosphomonoesterase activity present in the nucleotide pyrophosphatase preparations used. When the nucleotide pyrophosphatase digestion was followed by treatment with bacterial alkaline phosphatase, before electrophoresis, all the radioactivity from both peaks I and II migrated in a single spot identified as m7G (Fig. 2). Thus, in both the presumptive Y-terminal structures, methyl-labeled radioactivity is present exclusively in m'GMP. Since peaks I and II have the same methylated compound, they must differ either in the number of phosphates in the pyrophosphate linkage or in the other bases.

Number of Blocked 5'-Terminal
Phosphates-To determine the number of phosphate residues present in 'the PI-and bacterial alkaline phosphatase-resistant dinucleotides, material from peaks I and II ( Fig. la) Fig. 3, radioactivity from both peak I and peak II eluted in the same position, between marker nucleotides of charge -2 and -3. P,-and bacterial alkaline phosphatase-resistant dinucleotides having m7G and a net charge of -2.5 must have 3 phosphate residues, each phosphate contributing 1 negative charge and the m'G Yz a positive charge. Hence, both dinucleotides have a structure, m7G(5')pppN.
Their difference can only be in the identity of the unmethylated nucleosides, N.