RNA binding proteins and mRNA localisation in Drosophila sperm development

Jackson, Dana 2024. RNA binding proteins and mRNA localisation in Drosophila sperm development. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

In Drosophila melanogaster sperm development, male germline cells progress through a tightly regulated program of mass displacement, growth and proliferation. Each stage is defined by cell-type-specific changes to shape, volume and gene expression profiles. After meiosis, the newly developed spermatids undergo synchronised differentiation, elongation and maturation; transforming from round cells to highly specialised and polarised, needle-like cells that extend to 1.8 mm in length. Individualisation then separates these interconnected spermatids to yield mature, coiling sperm. While most spermatogenesis-specific transcriptional activity occurs in pre-meiotic spermatocytes, a small number of genes are also post-meiotically transcribed in the mid-to-late elongating spermatids – at a point preceding the histone-transition protein-protamine switch during chromatin reconfiguration. These genes express asymmetrically localised mRNAs that accumulate towards the tail-ends of growing spermatid cyst bundles in unusual localisation patterns of shooting speckled “comets” or U-shaped acorn “cups”. We know that this mRNA localisation precedes the formation of distinct protein gradients but, apart from the spatiotemporal profiles of their post-meiotic expression, very little is known about the molecular and regulatory driving forces that underpin this biological phenomenon. There are, however, several RNA binding proteins (RBPs) that are enriched in similar subcellular regions at the spermatid tail-ends. We therefore hypothesised that some of these RBPs are contributing to the post-transcriptional regulation of localised post-meiotic RNA transcripts in sperm development. RNA-affinity pull-down assays of 11 comet and cup mRNAs revealed differential binding of 4 RBPs in vitro, with the amount of interacting RBP varying depending on the mRNA of interest. The highly conserved IGF-II mRNA-binding protein (Imp) was the only RBP to bind all comet and cup transcripts in our test panel. The Imp protein family are key biological players, involved in the binding, transportation and post-transcriptional processing of various localised mRNAs,ncluding those that encode cell fate determinants and actin transcripts. In Drosophila sperm development, Imp is expressed at two spatiotemporally distinct pre-meiotic and post-meiotic phases, suggesting the possibility of pleiotropic functionality in the testis. While Imp is known to play an important role in the maintenance of male germline stem cells during early pre-meiotic sperm development, little is known about its activity throughout the latter stages (spermiogenesis) or its role within the elongating spermatids. As Imp demonstrated consistent binding to our panel of comet and cup mRNAs, is highly expressed and localised within the spermatid tail-ends, and is implicated in numerous developmental processes, we decided to take it forward as our main candidate for further investigation. We optimised the Cleavable-Affinity Purification (Cl-AP) to precipitate out whole, multi-protein Imp:mRNA ribonucleoprotein (RNP) complexes from pooled Drosophila testis homogenates. In doing so, follow-up comparative proteomics and RNA-Sequencing of the Cl-AP-purified, testis-specific Imp RNP complexes identified 29 Imp-enriched protein binding partners and 249 Imp-interacting mRNAs, respectively. Several were associated with RNA binding, actin regulation, microtubule dynamics and translational activities. We also performed a series of phenotypic analyses via RNA interference (RNAi) screening to elucidate the functional role of imp in the Drosophila testis and late sperm development. RNAi knockdowns of imp in vivo resulted in a variable spectrum of abnormal testis phenotypes, including mislocalisation of mRNA transcripts, downregulation of localised RNA and fluorescent protein reporter signals, and spermatid elongation defects. Overall, this suggested a context-dependent role of Imp in the post-meiotic stages of sperm development. Taken together, we have subsequently proposed a novel Imp-facilitated, F-actin-dependent anchoring and elongation mechanism that may regulate localised active transport and translation of post-meiotic mRNAs at the spermatid tail-ends.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	13 May 2025
Last Modified:	13 May 2025 18:09
URI:	https://orca.cardiff.ac.uk/id/eprint/178231

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