In the middle of sperm development, transcription ceases because of the onset of chromatin condensation and elongation in step 9 spermatids. Thus, proteins needed for subsequent seven steps of development (steps 10-16) into spermatozoa must be synthesized using mRNAs transcribed in earlier spermatogenic cells (e.g. late pachytene spermatocytes and round spermatids). These pre- synthesized mRNAs must be stabilized and stored in the subcytoplasmic domain called ribonucleoprotein particles (RNPs) for days before their translocation to the polyribosome for translation. The delayed translation is a typical post-transcriptional regulation and has been demonstrated to be essential for spermiogenesis. However, the underlying molecular mechanism remains elusive. Based upon the fact that mRNAs subject to delayed translation contain binding sites of both RNA-binding proteins (RBPs) and miRNAs in their 3'UTRs, we hypothesize that interactions between RBP (e.g., ELAVL1) and miRNA/miRISCs at the 3'UTRs determine the fate of their target mRNAs during spermiogenesis. We will use ELAVL1 as an example to test this hypothesis. First, we will identify all ELAVL1 target mRNAs and the positional relationship between ELAVL1 recognition elements and miRNA targeting sites in all ELAVL1 target mRNAs that display delayed translation during spermiogenesis (Aim 1). We will then determine the molecular composition of ELAVL1-based machineries that operate in the nucleus, in the RNP and polysome subcytoplasmic compartments to control the fate of ELAVL1 target mRNAs (Aim 2) using proteomic analyses. Finally, we will study how ELAVL1-miRNA interactions affect the fate of individual ELAVL1 target mRNAs through reporter assays in vitro and via generation of mutant mouse lines expressing ELAVL1 target mRNAs lacking the binding sites for either ELAVL1 or miRNAs in the 3'UTRs (Aim 3). We expect to uncover a critical molecular mechanism through which the mRNA fate is accurately controlled during spermiogenesis. Knowledge from this study will help the development of novel diagnostics and therapeutics for male infertility, as well as non-hormonal male contraceptives.
The proposed study will reveal the molecular mechanism through which transcription and translation of numerous genes essential for the last several steps of sperm formation get uncoupled. Data from this study will potentially lead to novel diagnostics and treatments for male infertility, and the development of novel non-hormonal male contraceptives.
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