Nucleotide sequences and mapping of novel heterogenous 5′-termini of adenovirus 2 early region 4 mRNA

Abstract

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.

Unusual heterogeneity of the 5′-termini of human adenovirus type 2 early region E2 mRNA

“…In previous studies, we have purified mRNA specific to each Ad2 early gene region and characterized the 5'-termini of the purified mRNA. We observed heterogeneous 5'-termini in EIA, E1B, and E4, and we were able to identify "major" termini from each region which we sequenced and mapped on the Ad2 DNA genome (13)(14)(15). Similar results were reported by Baker and Ziff (16).…”

“…1 were mixtures of oligonucleotides derived from authentic 5'-termini which have 5'-terminal 2'-O-methyl nucleotides (pNm) and from internal nicks which have 5'-terminal unmethylated nucleotide (pN). As we have previously shown (13)(14)(15), oligonucleotides containing methylated components (both 2'-0-methyl and base methylated oligonucleotides) do not migrate differently from unmethylated oligonucleotides by 2D-electrophoresis-homochromatography when the nucleotide sequence or composition is the same. We did not obtain major 5'-terminal oligonucleotides in our analysis of E2 mRNA purified by two different protocols, i.e.…”

“…Purified mRNA was digested in 10 pl of 20 mM sodium acetate, pH 5.3, containing 50 pg of carrier tRNA, with 2 pg of nuclease P1 (Boehringer Mannheim) at 370 for 2h, mixed with pNm markers (pA m,pm Am,pCmpUm,pGm, 0.2 A260 units each, rnm P-L Biochemicals), and applied to cellulose TLC plates (10 x 10 cm, EM Laboratories, Inc.). 2D-TLC was performed with isobutyric acid/0.5 N NH40H (5:3) in the first dimension and isopropyl alcohol/concentrated HCl/H20 (70:15:15) in the second dimension (15 (14). 32pAGp was prepared by digestion of 5'-32P-labeled RNA with RNase TI and purification by 2D-homochromatography (24).…”

“…f. Spot 27 is a mixture of pCmUmup and p(m6)AmUmUp, because (i) the 5'terminal nucleotides are pCm and p(m6)A ; (ii) the migation of PCm and p(m6)Am is almost the same in the first dimension (14); and (iii) the spot migrated faster than pAUp or pAGUp in the first dimension, thus the sequences must be more U-rich than p(m6)AMUp, p(m6)AmGIUp, pC'Up, pCMG'5p, which suggests that this spot contains trinucleotides.…”

“…Although resolution of pGM from pC in these plates is poor (designated by pC + NG ), the nucleotides detected in spots 8, 10, 16, and 22 are concluded to be pG because of their RNase A specificity. although some heterogeneous termini were also present (13)(14)(15). This suggests that the heterogeneity of the 5'-termini of E2 mRNA is much higher and of a different nature than that of other Ad2 early region mRNAs.…”