Graduate School of Life Science, Hokkaido University, Sapporo, Japan
Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto, Japan
Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo, Japan
Abstract Text: Many functional long noncoding RNAs (lncRNAs) are expressed in the mouse testis, but detailed molecular mechanisms of their action are poorly understood. We identified Tesra, a mouse testis-specific lncRNA, at the mouse Prss/Tessp locus and found that it is localized in both germ cells and Leydig cells. In this study, based on the knowledge that lncRNAs often regulate the expression of neighboring genes, we tested the possibility that Tesra regulates expression of the Prss/Tessp genes and investigated a molecular mechanism underlying it. We first performed the chromatin isolation by RNA purification (ChIRP) assay and found that Tesra bound to chromatin region of the Prss42/Tessp-2 promoter in germ cells from the testis. Moreover, the overexpression of Tesra increased the Prss42/Tessp-2 expression level and its promoter activity in the in vitro cell culture system. Thus, the Prss42/Tessp-2 gene, which is specifically expressed in spermatocytes, is likely to be a target of Tesra. To reveal a molecular mechanism underlying the regulation of the Prss42/Tessp-2 gene by Tesra, we searched for Tesra-binding proteins by the Ribotrap assay and the mass spectrometry analysis. One candidate protein, polypyrimidine tract binding protein 2 (PTBP2), was expressed in correlation with both Tesra and Prss42/Tessp-2 during testis development. Since both Tesra and PTBP2 are known to be localized in the nucleus of spermatocytes and our RNA immunoprecipitation (RIP) assay revealed the interaction between PTBP2 and Tesra in the nucleus of testicular germ cells, we next investigated the involvement of PTBP2 in the gene regulation by Tesra. We established the in vitro reporter gene assay system in which the expression of Tesra was induced by the Tet-on system and thereby, Prss42/Tessp-2 promoter activity was enhanced to drive the luciferase gene. The addition of doxycycline to this system induced the Tesra expression and increased luciferase activity, and the knockdown of PTBP2 significantly decreased luciferase activity. Lastly, we confirmed that PTBP2 bound to chromatin at the Prss42/Tessp-2 promoter in germ cells from the testis by the chromatin immunoprecipitation (ChIP) assay. Taken together, our results suggest that Tesra activates transcription of the Prss42/Tessp-2 gene in spermatocytes and that PTBP2 contributes to the Prss42/Tessp-2 transcriptional activation by Tesra. Because the Prss42/Tessp-2 gene is involved in the regulation of meiotic progression, Tesra may be an important regulator of spermatogenesis.
