FIGURE 2.

Analysis of the 3′ end of histone mRNAs and degradation intermediates using a standard primer or an oligoadenylated primer for cDNA synthesis. (A) The 3′ ends of the HIST2H2AA3 mRNA were determined by high-throughput sequencing using EnD-Seq. The positions of the last templated nucleotides are indicated, and the diversity of lengths of nontemplated tails is indicated by the different colors in the histogram. These data are the results from four independent experiments with RNA from S-phase cells. The y-axis gives the number of reads at each position, and the x-axis is the position of the last templated nucleotide. The sequence below the figure indicates the sequence of the processed histone H2a mRNA formed in the nucleus which is quantitatively trimmed on cytoplasmic histone mRNA. (B) Sequence of the 3′ end of the histone mRNA after processing. (C) Sequences of the most abundant 3′ ends of cytoplasmic RNA without any nontemplated nucleotides. (D) Sequences of the most abundant 3′ ends ending in 1-nt or 2-nt nontemplated tails. (E) Distribution of the nontemplated nucleotides in the 1- nt (top) and 2-nt (bottom) tails. (F,G) The sequences of the 3′ end of the H2AA3 mRNA from exponentially growing HeLa cells treated with hydroxyurea for 20 min as identified using the standard EnD-Seq method (F), showing the major 1- and 2-nt tails at the 3′ end, and longer tails in the 3′ side of the stem. The y-axis gives the number of reads at each position. After priming cDNA with an oligo(A)₃ tagged primer (G), the pattern of tails changes to reflect the distribution of molecules with tails of 3 nt or longer, and the number of these longer tails is dramatically increased since only oligouridylated histone mRNAs were sequenced. Note the pattern of tails in (G) is similar to the panel of >2-nt tails in (F). (H,I) The analysis of tails 5′ of the stem–loop from the same experiment in (F,G), with the 3′ ends of the RNAs determined by the standard EnD-Seq protocol (H) or the 3′ ends determined after priming cDNA with an oligo(A)₃ tagged primer (I). The position of the stop codon is indicated. The y-axis is the total number of reads at each position. (J) AppEnD analysis of nontemplated U-tails detected by priming cDNA with the 3A-primer. Note that the tail is identified correctly. (K) AppEnD analysis of sequences resulting from adventitious priming at an oligo(U) stretch in the RNA when cDNA was primed with the 3A-primer. These sequences are not identified as nontemplated tails by AppEnD, since we required the oligo(U) tails to be at least 3 nt long.