Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

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 times to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein pumilio 1 (Pum1) regulate the translation of target mRNAs and progression of oocyte maturation. Pum1 was shown to bind to Mad2 (also known as Mad2l1) and cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and cyclin B1 RNA granules in mouse oocytes. These Pum1 aggregates were dissolved prior to the translational activation of target mRNAs, possibly through phosphorylation. Stabilization of Pum1 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.

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.

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.