The alternative noncoding exons 1 of aromatase (Cyp19) gene modulate gene expression in a posttranscriptional manner

Abstract

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. Aromatase transcription is driven by multiple tissue-specific promoters, which result in the production of various mRNA transcripts that contain an alternative noncoding exon 1 followed by a common protein-coding region. Transcriptional activity of these promoters is the only known determinant for aromatase protein abundance in a given tissue or cellular context. To determine whether aromatase expression could be influenced by additional regulatory mechanisms, we used a common heterologous promoter to drive the expression of multiple aromatase cDNA sequences that differ only by the alternative exon 1 sequence. These expression vectors gave rise to vastly different levels of aromatase mRNA and protein in multiple cell lines examined. Furthermore, the relative abundance of several mRNA variants did not correlate with that of the corresponding protein product. The variation in mRNA and protein levels is most likely due to a negative effect of certain alternative exons 1 on RNA stability and protein translation. Deletional analyses indicate that the 5' regions of the adipose tissue-specific exons I.3 and I.4 contain the cis-acting elements responsible for modulation of aromatase levels. Thus, our work uncovers an important role of the alternative exons 1 in posttranscriptional regulation of aromatase gene expression.

Figure 1

Schematic diagram of various exon 1-containing aromatase cDNA constructs. Tissue-specific exon1 sequences were fused to the common aromatase coding sequence from exon 2 to the stop codon (dark shading). All cDNA clones are expressed from the CMV promoter-driven pcDNA3.0 vector. The sizes (in base pairs) of the first exons and the tissue specificity of their expression are indicated.

Figure 2

Expression levels of aromatase protein derived from aromatase mRNA variants. 293T, MCF-7, and KGN cells were transiently transfected with 2 μg constructs together with 50 ng GFP expression vector as an internal control. Twenty-four hours after transfection, Western blot analysis was performed using antibodies against aromatase and GFP. All results presented in this figure and the following were repeated at least three times.

Figure 3

Abundance of various exon 1-specific aromatase transcripts. Two micrograms of the indicated constructs were cotransfected with 50 ng GFP into 293T, MCF-7, and KGN cells. RNA was isolated 24 h after transfection and analyzed by real-time PCR. GFP was used for normalization.

Figure 4

In vitro transcription and translation of various exon 1-specific aromatase cDNA constructs. Ten percent of the final translation products were analyzed by 12.5% SDS-PAGE followed by autoradiography. Radiolabeled GST transcribed and translated in the same in vitro system was used as an internal control.

Figure 5

The 5′ region of exon I.4 is required for down-regulating the aromatase expression. A, A predicted secondary structure of exon I.4. Various base pair positions are indicated. B, Schematic representation of aromatase expression vectors with various deleted regions of exon I.4. C, Western blot analysis of aromatase protein derived from various I.4 deletion constructs. D, mRNA levels of various I.4 deletion transcripts as determined by real-time PCR.

Figure 6

The 5′ region of I.3 is both sufficient and important for repressing aromatase expression. A, A predicted secondary structure of exon I.3. B, Schematic diagram of various mutant I.3-containing constructs. The upstream AUG codons in exon I.3 were mutated individually (mt15 and mt46) or together (mt15 and -46). Asterisks and solid triangles represent wild-type and mutant sequences, respectively. The effect of the first 59 bp of exon I.3 on the aromatase protein (C) and mRNA (D) levels. Mutational effect of the upstream AUG codons on the aromatase protein (E) and mRNA (F) levels.