Discovery and Characterization of Novel Long Noncoding RNAs in Mouse Male Germ Cell Development

Abstract

男性不育問題影響全球數以百萬計家庭，而精子生成障礙乃其主因之一。精子生成是指從未分化的精原細胞先後進行兩次減數分裂分化為精母細胞及精子細胞，並最終產生成熟的精子的發育過程。這是動物繁殖後代的必要過程，且在雄性生殖年齡中持續進行。這一連串榦細胞樣祖細胞的分化過程，需在生殖細胞內多種分子精密地配合調控下，方能順利進行。因此，鑑別其中關鍵的調控基因不但可破解精子生成的分子基理，也可為不育治療及男性避孕提供可行靶點。可惜，科學界目前對精子發育中不同發展階段的基因調控仍未能徹底掌握。多年以來，關於轉錄組的研究大都局限於蛋白編碼基因，不過蛋白編碼基因只佔哺乳類動物轉錄組的大約百分之二，而更大部份卻屬於不會被轉譯為蛋白的非編碼核糖核酸 (RNA) 轉錄物。其中，包括微RNA、小干擾RNA、及與PIWI 蛋白相互作用RNA在內的小非編碼RNA 在生殖細胞發育中的功能已被廣泛研究及報道，然而長非編碼RNA 的角色卻鮮為人知。近期多項研究發現長非編碼RNA 在正常及疾病的發展過程中均十分重要，這為人類解開生殖細胞發育的分子機理提供了具突破意義的新線索。可是，長非編碼RNA 在精原榦細胞內發揮的作用仍屬未知。在本研究的第一部份，我們將透過分析高通量轉錄組數據得出的小鼠生殖細胞特異性長非編碼RNA 資料庫，並發現超過二千條在精子發育不同階段表達的新的長非編碼RNA。我們更建成了GermlncRNA 公共網上數據庫，以供全球研究員搜索、下載及分析這些數據。運用這些數據，我們發現名為Musashi-1 (Msi1) 的RNA 結合蛋白能優先結合到具精母細胞特異性的長非編碼RNA 上，並透過RNA 免疫沉澱證明之。進一步的功能實驗更顯示部份可與Msi1 結合的精母細胞特異性的長非編碼RNA 可以促進生殖細胞的增殖，並初步證明其機理可能是增強Msi1 對p21 及Erh 基因轉譯的調控作用。在本研究的第二部份，我們重新分析早前發表的基因芯片數據，並找出107 條表達量與小鼠精原幹細胞從六天到八個月的老化相關的長非編碼RNA。我們進一步解構這些與老化相關的長非編碼RNA，並透過與它們相關的蛋白編碼基因預測它們的潛在功能。總括而言，這項研究讓我們窺探到一個龐大而可能影響男性生殖細胞發展及老化的編碼–非編碼RNA 調控網絡，為增進理解此發育過程作出貢獻。Spermatogenic failure is a major cause of male infertility, which affects millions of couples worldwide. In sperm production, or spermatogenesis, undifferentiated spermatogonia differentiate into spermatocytes and spermatids through two rounds of meiotic division, and finally give rise to mature spermatozoa (commonly known as sperm). Success in this developmental cascade involves sophisticated regulations by various types of molecules in the developing germ cells. As a result, identifying critical regulatory genes controlling spermatogenesis provide the hints not only to understand the regulatory mechanism of spermatogenesis on molecular level, but also to identify potential targets for infertility management or contraceptives development. Despite the biological importance in male germ cell development, the underlying mechanisms of stage-specific gene regulation and cellular transition during spermatogenesis remain largely elusive.Previous genomic studies on transcriptome profiling were largely limited to protein-coding genes. Importantly, protein-coding genes only account for about 2% of mammalian transcriptome, majority are noncoding transcripts that do not translate to proteins. While small noncoding RNAs such as microRNAs, siRNAs, and Piwi-interacting RNAs are extensively investigated in male germ cell development, the role of long noncoding RNAs is relatively unexplored. Recent discoveries of long noncoding RNAs (lncRNAs) as critical regulators in various normal and disease development provided new clues for delineating the molecular regulation in male germ cell development. However, their functions in spermatogonial stem cell (SSC) biology remain a mystery.In Part I of this project, we provided a comprehensive mouse germ cell-specific lncRNA catalogue derived from high-throughput transcriptome data and discovered over 2,000 novel lncRNAs expressed at different stages of spermatogenesis. We also developed GermlncRNA, a public web-based database, for other researchers to search, download, and analyze the lncRNA data. Based on GermlncRNA data, we identified an Spga-specific RNA-binding protein Musashi-1 (Msi1), which preferentially interacted with Spga-specific lncRNAs (Spga-lncRNAs). The interaction between Msi1 and selected Spga-lncRNAs was validated by RNA immunoprecipitation experiment. Further functional studies demonstrated that some of these Msi1-bound Spga-lncRNAs function as activator for germ cell proliferation, probably through enhancing the Msi1 regulation on p21 and Erh translation.In Part II of this project, we re-analyzed our previously published microarray data to determine lncRNA expression during mouse SSC aging, and identified 107 aging-associated lncRNAs (Age-lncRNAs) showing aging-dependent expression trends in SSCs from 6-day to 8-month. We further characterized these Age-lncRNAs and predicted their potential functions by associating with protein-coding genes.Taken together, this study reveals an extensive coding-noncoding regulatory network which may function behind germ cell development and aging.Luk, Chun Shui.Thesis Ph.D. Chinese University of Hong Kong 2015.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only