PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene.

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells.

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.

A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis†.

The progression of spermatogenesis is precisely controlled by meiotic stage-specific genes, but the molecular mechanism for activation of such genes is still elusive. Here we found a novel testis-specific long noncoding RNA (lncRNA), Tesra, that was specifically expressed in the mouse testis at the Prss/Tessp gene cluster on chromosome 9. Tesra was transcribed downstream of Prss44/Tessp-4, starting within the gene, as a 4435-nucleotide transcript and developmentally activated at a stage similar to that for Prss/Tessp genes. By in situ hybridization, Tesra was found to be localized in and around germ cells and Leydig cells, being consistent with biochemical data showing its existence in cytoplasmic, nuclear, and extracellular fractions. Based on the finding of more signals in nuclei of pachytene spermatocytes, we explored the possibility that Tesra plays a role in transcriptional activation of Prss/Tessp genes. By a ChIRP assay, the Tesra transcript was found to bind to the Prss42/Tessp-2 promoter region in testicular germ cells, and transient overexpression of Tesra significantly activated endogenous Prss42/Tessp-2 expression and increased Prss42/Tessp-2 promoter activity in a reporter construct. These findings suggest that Tesra activates the Prss42/Tessp-2 gene by binding to the promoter. Finally, we investigated whether Tesra co-functioned with enhancers adjacent to another lncRNA, lncRNA-HSVIII. In the Tet-on system, Tesra transcription significantly increased activity of one enhancer, but Tesra and the enhancer were not interdependent. Collectively, our results proposed a potential function of an lncRNA, Tesra, in transcriptional activation and suggest a novel relationship between an lncRNA and an enhancer.

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes.

BACKGROUND: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. RESULTS: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. CONCLUSIONS: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

Catalytic Synthesis of Saturated Oxygen Heterocycles by Hydrofunctionalization of Unactivated Olefins: Unprotected and Protected Strategies.

A mild, general, and functional group tolerant intramolecular hydroalkoxylation and hydroacyloxylation of unactivated olefins using a Co(salen) complex, an N-fluoropyridinium salt, and a disiloxane reagent is described. This reaction was carried out at room temperature and afforded five- and six-membered oxygen heterocyclic compounds, such as cyclic ethers and lactones. The Co complex was optimized for previously rare medium ring formation by hydrofunctionalization of unactivated olefins. The powerful Co catalyst system also enables the deprotective hydroalkoxylation of O-protected alkenyl alcohol and hydroacyloxylation of alkenyl ester to afford cyclic ethers and lactones directly. The substrate scope and mechanistic proof of deprotection were investigated. The experimental evidence supports the concerted transition state of the bond-forming step involving a cationic Co complex.

A genomic region transcribed into a long noncoding RNA interacts with the Prss42/Tessp-2 promoter in spermatocytes during mouse spermatogenesis, and its flanking sequences can function as enhancers.

Spermatogenesis is regulated by many meiotic stage-specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2-F3, and the three genes at this site (Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testis-specific lncRNA, lncRNA-HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp-2 gene. lncRNA-HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage-X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA-HSVIII locus specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. A 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity using a reporter-gene assay, yet this construct did not change lncRNA-HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstream and downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA-HSVIII locus with the Prss42/Tessp-2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp-2 gene. Mol. Reprod. Dev. 83: 541-557, 2016. © 2016 Wiley Periodicals, Inc.