Evaluation of coding-independent functions of the transcribed bovine aromatase pseudogene CYP19P1
© Chwalisz and Fürbass; licensee BioMed Central Ltd. 2014
Received: 30 September 2013
Accepted: 13 June 2014
Published: 20 June 2014
CYP19A1 encodes the aromatase which catalyzes the final reaction of estrogen biosynthesis. The bovine genome also contains a non-coding copy of CYP19A1, the transcribed pseudogene CYP19P1. Whereas CYP19A1 is transcribed in all estrogen-producing tissues, mainly in the placenta and gonads, the CYP19P1 transcript so far was detected in the placenta. Strikingly, one sequence segment of both transcripts exhibits an exceptional high identity of 98%, which implies selective pressure and suggests some kind of function. Only recently, indeed, coding-independent functions of several transcribed pseudogenes were reported. Therefore, we analyzed CYP19P1 and CYP19A1 transcripts with the aim to detect clues for gene–pseudogene interference.
The CYP19P1 transcript was first examined in silico for the presence of microRNA coding sequences and microRNA targets. Further, to identify tissues where CYP19P1 and CYP19A1 transcripts are co-expressed, as a pre-requisite for transcript interference, expression profiling was performed in a variety of bovine tissues. Our in silico analyses did neither reveal potential microRNA coding sequences, nor microRNA targets. Co-expression of the CYP19 loci was demonstrated in placental cotyledons and granulosa cells of dominant follicles. However, in granulosa cells of dominant follicles the concentration of CYP19P1 mRNA was very low compared to CYP19A1 mRNA.
CYP19P1 and CYP19A1 transcripts might interfere in placental cotyledons. However, in granulosa cells of dominant follicles relevant interference between gene and pseudogene transcripts is unlikely to occur because of the very low CYP19P1/CYP19A1 transcript ratio.
CYP19A1 encodes the aromatase which catalyzes the conversion of androgens to estrogens. During an earlier screening of a bovine placental cDNA library for CYP19A1 clones, we also isolated cDNA clones of a homologous pseudogene, CYP19P1[1, 2]. Gene and pseudogene transcripts exhibit major differences due to the loss of several exons in the CYP19P1 transcript. Furthermore, numerous mutations in the pseudogene sequence produced multiple translational stop codons in all reading frames and thereby abrogated its protein-coding function. Strikingly, however, mutations are unevenly distributed. A sequence segment of 177 bp corresponding to exon 5 of CYP19A1 is highly conserved, showing 98% identity, compared to 89% sequence identity in general. Both CYP19 loci are located on the same strand of chromosome 10, being separated by 20 kb of genomic DNA[3, 4].
Pseudogenes for long were considered as defunct copies of functional genes. However, recent evidence suggests that some pseudogenes might exert coding-independent functions[5, 6]. Interestingly, gene-pseudogene interference was demonstrated by selective knock-down of the ABCC6P1 pseudogene, which led to a decreased transcription of its ABCC6 parent gene. This recent evidence prompted us to take up again our analysis of the CYP19P1 pseudogene with the aim to detect clues of gene-pseudogene interference. To this end, transcripts were searched in silico for microRNA-coding sequences and microRNA targets. Further, expression profiles of CYP19A1 and CYP19P1 were analyzed in a variety of bovine tissues.
Materials and methods
Samples from placentas, ovarian granulosa cells, fetal ovaries, endometria, adrenal glands and livers were collected from slaughtered cows in a local abattoir. Tissue samples were stored in RNAlater (Qiagen, Hilden, Germany) at -20°C. Granulosa cells were frozen in liquid nitrogen and stored at -80°C. Placental cotyledons and caruncles were separated manually. Despite careful separation, caruncle samples might contain traces of cotyledonary cells. Ovarian dominant and pre-ovulatory follicles collected before and after the LH-surge, respectively, were identified as described in. Follicles were punctured with 18G needles and granulosa cells were aspirated. Total RNA was prepared using the NucleoSpin RNA II Kit (Macherey-Nagel, Düren, Germany), according to the supplier´s protocol. This procedure included on-column DNaseI digestion to remove traces of DNA. RNA was quantified in a NanoDrop 1000 spectrophotometer (PeQLab, Erlangen, Germany). RNA integrity was confirmed by denaturing agarose gel electrophoresis.
The statistical analyses were performed with the Sigma Plot 12.0 Analysis System (Jandel Scientific, San Raffael, CA, USA).
Results and discussion
The remarkably high conservation of CYP19P1 in the exon 5-homologous sequence segment (henceforth referred to as P1-exon 5) implies selective pressure. Hence, it might well be that the pseudogene exerts a coding-independent biological activity via the P1-exon 5. Because pseudogene-derived small interfering RNAs were shown to regulate gene expression in mouse oocytes, we examined if also the CYP19P1 transcript encodes microRNAs. We performed in silico analyses of the P1-exon 5 using the free software RNAfold to detect putative stable pre-microRNA hairpins. However, no such structures were predicted by the program. The cellular abundance of the tumor suppressor gene PTEN transcript was found to be regulated by the transcript of the highly homologous PTENP1 pseudogene via competition for microRNA binding. To evaluate if the CYP19 gene pair could also interfere this way, we searched the P1-exon 5 for microRNA target sites using the MIRANDA software. However, probable targets of known microRNAs were not found. Other pseudogenes are transcribed in an antisense orientation and lead to silencing of their parent genes by translational interference. However, this mode of action can not apply to CYP19P1 and CYP19A1 which are both encoded by the same strand of chromosome 10 and hence are transcribed in the same orientation.
We thank Dr. R. M. Brunner and F. Hadlich for providing assistance with in silico analyses, Maren Anders and Veronica Schreiter for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG).
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