Naturally occurring antisense RNA of histone H2a in mouse cultured cell lines

Abstract

Background: An antisense transcript of histone H2a that has no significant protein-coding region has been cloned from a mouse full-length cDNA library. In the present study, we evaluated this transcript by using RT-PCR and compared the expression patterns of the sense and antisense transcripts by using quantitative RT-PCR (qRT-PCR).

Results: This antisense RNA was expressed in three mouse cell lines. We call it ASH2a. ASH2a includes not only the complementary sequence of the transcript of Hist2h2aa2 (a replication-dependent histone H2a gene), but also that of the promoter of Hist2h2aa2. The upstream genomic sequence of the transcription start site of the ASH2a-coding gene (ASH2a) lacks both CCAAT and TATA boxes. This absence suggests that the regulation of ASH2a is different from that of the replication-dependent histone H2a genes. Findings from qRT-PCR indicated that the expression pattern of ASH2a was different from that of Hist2h2aa2. Expression of Hist2h2aa2 peaked at 2 to 4 h during S-phase, but that of ASH2a peaked at 1 h.

Conclusion: We showed the existence of ASH2a, a histone H2a antisense RNA, in mouse cultured cells. The expression pattern of ASH2a is different from that of the sense RNA.

Figure 1

(a) Nucleotide sequence of ASH2a. ASH2a is encoded from positions 1 to 2427. Bold characters indicate overlap with the Hist2h2aa2 transcript (italic = protein-coding region). Arrows indicate primers used in this study. (b) Relationship between Hist2h2aa2 and ASH2a RNAs. Arrows indicate the locations of the primers.

Figure 2

(a) RT-PCR products from cDNAs obtained by priming total RNA with random hexamers. Lanes: 1, DNA ladder (100-bp ladder, TOYOBO); 2–7, RT-PCR products amplified with primers F1 and R1 (upper) and those amplified with primers F1 and R2 (lower). RNA was extracted from Hepa 1–6 (lanes 2 and 5), 3T3 (lanes 3 and 6), and LLC (lanes 4 and 7) cells. Superscript III was not added in the reaction of lanes 2–4. Lane 8, PCR product of genomic DNA amplified with primers F1 and R1 (upper) and that amplified with primers F1 and R2 (lower). Arrows indicate the expected products. (b) Patterns of digestion of PCR products by PstI. Lanes: 1, DNA ladder; 2–4, digests of PCR products amplified with primers F1 and R1. PCR product was produced from Hepa 1–6 (lane 2), 3T3 (lane 3), and LLC (lane 4). Lanes 5–7, digests of PCR products amplified with primers F1 and R2. PCR product was produced from Hepa 1–6 (lane 5), 3T3 (lane 6), and LLC (lane 7). Arrows indicate the expected products. (c) RT-PCR products and the EcoRI-digest patterns of cDNAs obtained by priming total RNA with the specific primer R3. Lanes: 1 and 8, DNA ladder; 2–7, RT-PCR products amplified with primers F2 and R3. RNA was extracted from Hepa 1–6 (lanes 2 and 5), 3T3 (lanes 3 and 6), and LLC (lanes 4 and 7). Superscript III was not added in the reaction of lanes 2–4. Lanes 9–11, EcoRI-digests of PCR products amplified with primers F2 and R3. PCR product was produced from Hepa 1–6 (lane 9), 3T3 (lane 10), and LLC (lane 11). Arrows indicate the expected products.

Figure 3

Representative amplification plot. Curves indicate amplification from transcripts of GAPDH (first group of rising curves), Hist2h2aa2 (second), and ASH2a (third). Different colors indicate that each result from 0 h to 12 h (13 points). X-axis, cycle numbers; Y-axis, ΔRn.

Figure 4

Transcript expression patterns. (a) Transcripts of Hist2h2aa2. (b) Transcripts of ASH2a. X-axis, time (hours); Y-axis, relative expression level, adjusted to 1.0 at 0 h. The qRT-PCR analyses were performed 4 times (indicated by different colors).