- Title
- A focus on the essential roles of the Musashi family of RNA binding proteins during mammalian spermatogenesis
- Creator
- Sutherland, Jessie Maree
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2015
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Spermatogenesis describes the complex process male gamete development whereby spermatogonial stem cells undergo a series of mitotic amplification, meiotic divisions, and a series of profound morphological changes in order to produce physically mature spermatozoa This self-renewing process requires the appropriate translational programming of more than 700 mRNAs. It is therefore an interesting feature of spermatogenesis that despite the cells requirements for continued growth and rapid proliferation, it undergoes extended periods of transcriptional quiescence. As such, spermatogenesis is highly reliant on mechanisms of post-transcriptional regulation, driven by RNA binding proteins which remain abundantly expressed throughout this process. The Musashi family of RNA binding proteins are historically renowned for their fundamental roles in stem cell function, cell fate determination, CNS development, cellular proliferation, and tumorigenesis. Comprising of two mammalian orthologues, Musashi-1 (MSI1) and Musashi-2 (MSI2), this family of proteins remains highly conserved across vertebrate species. Given the established importance of Musashi in stem cells, it was surprising that these proteins had not been previously explored in the stem cell niche of the testis during spermatogenesis and we hypothesised a role for Musashi in this process. From pioneering studies we discovered an essential function for Musashi as critical regulator of testis germ cell maintenance and meiosis via genetic screening in Drosophila, and more recently have identified the differential expression of MSI1 and MSI2 in the mammalian testis. The purpose of this thesis was to describe and characterise the expression and function of both MSI1 and MSI2 during mammalian spermatogenesis, primarily using a mouse model. Through examining the differential expression of mammalian MSI1 and MSI2 during germ cell development, we found that MSI1 was predominately localized in mitotic gonocytes and spermatogonia, while MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Examination of the role of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell specific over-expression of full-length isoforms of Msi1 (TgMsi1) or Msi2 (TgMsi2). These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with the Msi2 over-expression resulting in male sterility. Focussing further on the differential expression of MSI1, in particular the transition between cytoplasmic and nuclear localisation, we identified two unique RNA-binding targets of MSI1 in spermatogonia in Msi2 and Erh. Again utilising our TgMsi1 animal model we demonstrated potential roles for MSI1 in translational regulation, providing evidence to suggest that nuclear import protein IPO5 facilitates nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. In terms of defining the role MSI2, differential gene expression studies and comparative protein expression analyses aided in the identification key molecular networks, and essential biological processes, globally affected by overexpression of Msi2. Functional analysis revealed Tbx1 and Piwil1 as direct RNA-binding targets of MSI2-mediated translational repression. Protein-protein interaction studies revealed MSI2 acts in complex with splicing factor SFPQ in testis germ cells, suggesting a function for MSI2 in pre-mRNA processing Overall this body of work provides strong evidence for the essential roles and unique functions of the Musashi family of RNA binding proteins during spermatogenesis, firmly establishing clear evidence for both proteins in post-transcriptional gene regulation. For MSI1 as a master regulator of translation repression during early germ cell development; and MSI2 as a key regulator of later stage male gamete development, involved in pre-mRNA processing.
- Subject
- spermatogenesis; RNA binding proteins; Musashi family; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1313463
- Identifier
- uon:22598
- Rights
- Copyright 2015 Jessie Maree Sutherland
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Abstract | 558 KB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Thesis | 10 MB | Adobe Acrobat PDF | View Details Download |