Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate timings to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein Pumilio1 regulate the translation of target mRNAs and progression of oocyte maturation. Pumilio1 was shown to bind to Mad2 and Cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and Cyclin B1 RNA granules in mouse oocytes. These Pumilio1 aggregates were dissolved prior to the translational activation of target mRNAs possibly by phosphorylation. Stabilization of Pumilio1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.

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.

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BackgroundSubcellular 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.ResultsThe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0071-z) contains supplementary material, which is available to authorized users.

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.