J. gen. ViroL (I975), 27, 247-z5o 247 Printed in Great Britain Location and Abundance of Poly (A) Sequences in Sendai Virus Messenger RNA Molecules (Accepted 14 January I975) SUMMARY Adenine-rich sequences from i8 S Sendai virus messenger RNA species were 99 % adenylate, 3'-OH terminal, and were present in at least 50 % of the RNA molecules. Intact virus messenger RNA molecules were resistant to exonucleolytic attack by polynucleotide phosphorylase, suggesting that their 3'-termini are masked. Most messenger RNAs of eukaryotic cells and their viruses contain adenine-rich sequences (Weinberg, r973). Sendai virus, a paramyxovirus, is no exception. It specifies messenger RNAs which are complementary in base sequences to virus particle RNA; most, if not all, of these RNAs sediment at about ISS (Kingsbury, I973); they contain adenine-rich sequences which sediment at about 4S (Pridgen & Kingsbury, 1972). We now report more details about the structure of these adenine-rich sequences. In all experiments, RNA was obtained from chick embryo lung (CEL) cells which were infected with Io p.f.u, of low multiplicity passage Sendai virus per cell and incubated at 37 °C. At 48 h after infection, when virus production was at its peak, cells were treated for I h with 50 #g of actinomycin D per ml. A radioactive precursor of RNA was then added and incubation at 37 °C was extended in the presence of actinomycin D for 4 h. Cytoplasmic extracts were made by the method of Penman (I966) and RNA was isolated from them with SDS and phenol (Pridgen & Kingsbury, I97z). The extracts were centrifuged in sucrose velocity gradients and RNA sedimenting at I8 S was selected. This RNA sedimented again as a single peak at I8S when it was boiled for 3 min in o.oi M-tris HC1, pH 7"4, rapidly cooled, and recentrifuged. All enzymes used were products of Worthington Biochemical Corporation, and were the purest available. About Io % of p2P]-labelled ISS virus RNA was resistant to a mixture of bovine pancreatic and T1 ribonucleases. Infected cells were labelled with 250 #Ci of carrier-free [3aPO~-] per ml of medium compounded without orthophosphate but containing 5o/zg of actinomycin D/ml. The 18 S RNA was digested for 30 rain at 24 °C with 50 #g of pancreatic ribonuclease and 1/zg of T1 ribonuclease per ml of 0'3 M-NaC1, o-ooi M-EDTA, o'oo5 Mtris-HC1 (pH 7"4). The digest was passed through a Sephadex G5o column equilibrated with the same buffer containing 7 M-urea. Material which emerged in the void volume was hydrolysed with alkali and the ribonucleotides were separated and identified (East, 1968). The nucleotide composition of this ribonuclease-resistant material was 99 % adenylate (Table 1), whereas the I8 S RNA itself contained about equal amounts of [32p] in each of the four ribonucleotides (data not shown). In view of its base composition, we will refer hereafter to the ribonuclease-resistant, adenine-rich fragment as the 'poly (A)' portion of Sendal virus messenger RNA. We now present evidence which shows that this poly (A) is located at the 3' terminal position in the Sendai virus messenger RNAs, in common with other virus and cellular messenger RNAs 248 Short communications Table L Base composition of the ribonuelease-res&tant portion of Sendai virus 18 S messenger RNA Mol ~* f Adenine Uracil Cytosine Guanine 99'3 (+o'5) o.I (_+o.I) o'3 (___o'3) o'3 (_+o.z) * The numbers in parentheses are the standard deviationsof the means of Io determinations. (Weinberg, I973). The poly (A) obtained by combined T1 and pancreatic ribonuclease digestion of [3HI-adenine-labelled I8S RNA was treated with Micrococcus tuteus polynucleotide phosphorylase under conditions where the phosphorylase acts as a 3'-OH terminal exonuclease (Sheldon et al. I97zb ). At an enzyme concentration of o.6 units/ml in o-oo5 M-MgC12, o.oi M-sodium phosphate, o.I M-tris-HC1 (pH 8"5), the poly (A) was 90 ~ digested in zo min and completely digested in 40 min at 37 °C. This experiment was controlled in several ways. The rate of enzymatic degradation did not increase after pre-treatment of the poly (A) with Escherichia coli alkaline phosphatase. The polynucleotide phosphorylase itself was free of contaminating phosphatase by two criteria: it was inactive against p-nitrophenyl phosphate (Garen & Levinthal, I96o) and it digested no more than 40 ~ of a commercial poly (A) preparation that had been incubated briefly with pancreatic ribonuclease at low ionic strength to generate fragments bearing both 3'-and z'-terminal phosphate. Thus, it was established that most of the poly (A) segments from Sendai virus I8S messenger RNA terminated in native 3'-OH groups. Attempts to digest intact Sendai virus I8S RNAs with polynucleotide phosphorylase were unsuccessful (Fig. I). [~H]-adenine labelled RNA was incubated at 37 °C with o-6 units of polynucleotide phosphorylase per ml, as described before. At intervals, samples were placed in ice-cold o'oi25 M-EDTA, o.I 5 M-NaC1 to stop the digestion, and either precipitated with 5 ~ trichloroacetic acid or ribonuclease-treated and then acid precipitated. It can be seen that neither intact 18 S mRNA nor the poly (A) segment derived from it by ribonuclease treatment was affected by polynucleotide phosphorylase under these conditions. This was unexpected in view of the ease with which other messenger RNAs and our nuclease-released poly (A) were digested (Sheldon et al. ~97ab; Williamson, Crossley & Humphries, I974). A simple explanation is that intramolecular secondary structure blocks the 3'-termini of the intact Sendal virus messenger RNAs. In an attempt to expose 3'-termini by denaturing the RNAs they were boiled in water and cooled rapidly before adding the phosphorylase. The result was the same; no enzymatic degradation occurred, indicating that the 3'-termini became blocked again. However, a more drastic treatment was effective. Another 18 S mRNA preparation was treated with o'3 M-KOH for I rain at 23 °C and neutralized by passage through a small Dowex 5o (H +) column before treatment with phosphorylase (Fig. I). About t6 ~ of the alkali-treated ~8S mRNA was digested by the enzyme and the poly (A) portion was more rapidly and more extensively digested, confirming that it was in a 3' terminal position. Longer or shorter alkali treatments reduced the amount of I8 S RNA digested by the enzyme. Presumably, shorter treatments cleaved fewer molecules, releasing fewer free 3'-OH terminal fragments, whereas longer treatments reduced the lengths of these fragments. Although the evidence indicates that 3'-termini are blocked in intact Sendal virus Short eommtmications 249 L3 ~9 70 ,5 60 50 I S . _ I 10 _ I 15 Time (rain) Fig. I. PolynucIeotide phosphoryiase treatraent of Sendal virus I8S messenger RNA. (3--(3, ~SSmRNA; O--O, poly (A) derived from ISSmRNA; 72--f2, alkali-treated 18SmRNA; lilt--II, poly (A) derived from alkali-treme6 1 ~ mP.N~k. messenger RNAs ur~der tb~ese conditions, the relevance of this 5rtding t~3 the in vivo conformations or functions of these RNAs is rtot clear. In other experiments we measured the proportion of Sendal virus ~8S messenger RNAs which contained poly (A) using polynueleotide binding methods. From 60 to 85 o/~ of the RNA was bound by poly (U) f~lters (St~eld~n, ]urale, & Kales ~97za) irt different experiments, whereas about 50 ~o bound eitb~er to unmodified cellulose (Kitos, Saxon & Amos, t972 ) or to poly (U)-Sepharose (Lindberg & Persso~, ~9"/Z). The RNA had been labelled with [ZH]-adenine in the latter two cases, so that ribonuclease treatment could be used to estimate the poly (A) content of the hound and unbound material. The unboond RNA was less than Io ~o ribonuclease-resistant whereas the bound RNA was more than zo ~/o resistant to ribonuclease after elution from the columns. Poly (A) segments are heterogeneous in length (Weinberg, ~973) and it has been sho~tt that different polynucleotide binding procedures have different se}ectivities (Gorski et aL ~974). Thus, at least ttalf of Set~dai virus I8S RNA molecules contain poly (A) sequences long enough to bind to cellulose or to poly (U)-Sepharose. The remainder are relatively deficient in poly (A). The RNA molecules deficient in po~y (A) a~e ~ t celVspecified messenger RNAs or slowly sedimenting virus-specific RNAs of the virus particle type. Actin(~mycirt D eliminates the former (Blair & Robinson, ~968) and lo,~-mu~tip[ictty passage of virus prevents the emergence of defective-interfering virus particles mhich generate the tatter (Kingsbury & Portner, I97o). Similar data have been obtained with vesicular stomatitis virus (Soria & Ftuang, 1973), 250 Short communications another 'negative-strand virus' (Baltimore, I97 0. Here, too, a significant portion of RNA molecules which qualify as virus messenger RNA by virtue of sizes and base sequences are deficient in poly (A). Some insight into the function of poly (A) may be gained by determining whether these poly (A)-deficient RNAs can act as templates for virus proteins and whether they originate as such or are derived from poly (A)-rich congeners. This work was supported by USPHS Research Grant AI-o5343, USPHS Childhood Cancer Research Center Grant CA-o848o, USPHS Training Grant CA-o5~76, ALSAC, and USPHS Career Development Award HD-I4,49I to D.W.K. 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