Estrogen down-regulation of the Scx gene is mediated by the opposing strand-overlapping gene Bop1

Abstract

Recent genome-wide transcriptome studies suggest the presence of numerous bidirectional overlapping coding gene pairs in mammalian genomes. Various antisense RNAs are reported as non-coding RNAs that regulate the expression of sense RNA. However, it is still unclear whether the expression of bidirectional overlapping coding genes are regulated by the opposite strand gene transcript acting as a non-coding RNA. Bop1 and Scx are a pair of bidirectional overlapping coding genes related to cellular proliferation and differentiation, respectively. Scx gene is localized in the intron 3 region of the Bop1 gene. The expression of these genes is reciprocally regulated by estrogen (E2) in the mouse uterus. In situ hybridization indicated that both genes are expressed in the uterine endometrial epithelial cells and that the antisense RNA of Scx (Bop1 intronic RNA) accumulates as a stable RNA in these cells. The existence of Bop1 intronic RNA was confirmed by reverse transcription-PCR and was increased after E2 treatment, coinciding with a decrease in Scx mRNA. Murine myoblasts expressing doxycycline-inducible endogenous Bop1 gene showed an increase in Bop1 intronic RNA and a simultaneous decrease in Scx mRNA. Murine fibroblasts expressing Scx mRNA from an exogenous Scx mini-gene indicated that the accumulation of Bop1 intronic RNA impairs the Scx gene expression in a trans-acting manner, which resulted in the reduction of the Scx mRNA level. This study demonstrates a novel example of hormone-stimulated intronic non-coding RNA down-regulating the expression of an opposing strand-overlapping coding gene.

FIGURE 1.

Schematic illustration of the Bop1 and Scx genes. The location of the Bop1 (red) and Scx (green) genes on mouse chromosome 15 is illustrated with their exon-intron structure. The boxes and lines indicate exonic and intronic regions, respectively. The arrowheads depict the direction of gene transcription. The positions of the probes for ISH are indicated as black boxes (Bop1 ISH and Scx ISH). The positions of PCR-amplified element are indicated as A, B, and C. Primer sets A and B are located on Bop1 intron 3. Set A does not overlap, and set B overlaps the Scx gene. Primer set C is located on Bop1 intron 2. Ex1, Ex2, Ex3, and Ex4–16 indicate exons 1, 2, 3, and 4–16, respectively.

FIGURE 2.

Expression of the Bop1 and Scx genes in the E2-treated OVX mouse uterus. Animals (six or four mice per group) were administered E2 with (dashed line) or without (solid line) ICI, and uterine total RNA was prepared at the times indicated. A, the mRNA levels of Bop1 (upper panel) and Scx (lower panel) gene were quantified by real-time PCR as described under “Experimental Procedures.” Results were normalized to the level of 18 S in each sample. The values are the means ± S.D. *, p < 0.01 compared with time 0. B, the result expressed as the log 10 ratio for changes relative to time 0 to compare the changes between Bop1 and Scx mRNA levels.

FIGURE 3.

Antisense RNA for the Scx gene transcript exists in the OVX mouse uterus. A, ISH results from serial sections of the OVX mouse uterus. ISH was performed using an antisense probe (AS) for Scx cDNA (panels a and b), a sense probe (S) for Scx cDNA (panels c and d), an antisense probe for Bop1 cDNA (panels e and f), and a sense probe for Bop1 cDNA (panels g and h). The position of the probes is indicated in Fig. 1. A nonspecific plant sequence was used as the negative control (panels i and j) as described under “Experimental Procedures.” The purple signal shows specific hybridization of the indicated probes. The boxes in panels a, c, e, g, and i are increased in magnification correspond to panels b, d, f, h, and j, respectively. Ep indicates endometrial epithelial cells, and Lu indicates lumen. Scale bars = 200 μm in panels a, c, e, g, and i and 50 μm in b, d, f, h, and j. B, total uterine RNA was prepared from the E2-treated mice at the indicated times. A representative RT-PCR result of various primer sets (A, B, C, Scx, Bop1, and 18 S) using the identical samples is shown. A, B, and C indicate the Bop1 intronic elements in Fig. 1. The samples of lanes 1–5 were reverse-transcribed from RNA extracted from E2-treated uteri at the indicated times (+RT). The samples of lanes 1′–5′ were not reverse-transcribed using the same RNA of 1–5 (−RT). Ten ng of genomic DNA were used as the positive control for the intronic RNA. C, E2 was administered to mice (six mice per group) with (dashed line) or without (solid line) ICI, and uterine total RNA was prepared at the times indicated. Real-time PCR was performed, and the results were normalized to the level of 18 S in each sample. The values are the means ± S.D. *, p < 0.01 when compared with time 0, **, p < 0.02 when compared with time 0.

FIGURE 4.

The down-regulation of Scx mRNA is correlated with the induction of endogenous Bop1 gene expression. A, Tet-On-Myc-C2C12 cells were treated with (+) or without (−) doxycycline, and total RNA was prepared 6 h after treatment. Real-time PCR was performed, and the results were normalized to the level of 18 S in each sample. The values are the means ± S.D. for four independent replicates. *, p < 0.01 when compared with (−). B, a representative RT-PCR result of various primer sets using the identical samples is shown. The positions of PCR-amplified Bop1 intronic elements A, B, and C are indicated in Fig. 1. The samples of lanes 1–4 were reverse-transcribed (+RT) from the RNA that was extracted from cells treated with (+) doxycycline (2 h) without (−) doxycycline. The samples of lanes 1′–4′ were not reverse-transcribed (−RT) using the same RNA as lanes 1–4. Ten ng of genomic DNA were used for positive control of intronic RNA. C, total RNA was prepared at 2 h after with (+) or without (−) doxycycline treatment. D, Tet-On-mBop1cDNA-C2C12 cells were treated with (+) or without (−) doxycycline, and total RNA was prepared 6 h after treatment. Real-time PCR was performed, and the results were normalized to the level of 18 S in each sample. The values are the means ± S.D. for four independent replicates. *, p < 0.01 when compared with (−), **, p < 0.05 when compared with (−).

FIGURE 5.

Scx mRNA expressed from the exogenous Scx genomic DNA is reduced by increasing the endogenous Bop1 gene expression. A, schematic representation of CMV-Scx-gDNA(+1), CMV-Scx-gDNA(−250), and CMV-Scx-cDNA is shown. The position of PCR primers used for the amplification of Scx mRNAs is indicated. The expected size of the PCR amplicons is shown to the right of each primer pair. Ex1 and Ex2 indicate exons 1 and 2, respectively. B, a representative RT-PCR result of Scx transcript is shown. The samples were prepared from BALB/3T3 cell lines containing CMV-Scx-gDNA(+1), CMV-Scx-gDNA(−250), CMV-Scx-cDNA, or pcDNA3 (as a negative control). The samples in lanes 2–5 were reverse-transcribed (+RT) from the RNA, and the samples of lanes 6–9 were not reverse-transcribed (−RT) using the same RNA of 2–5. MW, molecular weight markers. C, total RNA was prepared from treated (high serum medium; +) or untreated (low serum medium; −) CMV-Scx-gDNA(+1), CMV-Scx-gDNA(−250), and CMV-Scx-cDNA BALB/3T3 cells as described under “Experimental Procedures.” A representative real-time PCR result of Scx mRNA is shown. D, a representative real-time PCR result of Bop1 mRNA is shown. E, a representative real-time PCR result of Bop1 intronic RNA (primer set A) is shown. The RNA level was normalized to the level of 18 S in each sample. The values are the means ± S.D. for three independent replicates. *, p < 0.01 when compared with (−), **, p < 0.05 when compared with (−).