Characterization of target genes co-regulated by SMG6 and PIWIL1

Sperm counts are declining at an accelerated rate globally. Therefore, elusive studies need to be conducted to be able to understand spermatogenesis in detail, and thereby, find solutions to maintain male reproductive health. Telomerase-binding protein EST1A (SMG6) and P-element Induced Wimpy-Testis-Like Protein 1 (PIWIL1) are found in the chromatoid body (CB) which is a membrane-less organelle in the cytoplasm of male germ cells. The CB is crucial for RNA regulation during spermatogenesis. SMG6 is an endonuclease in the Nonsense-Mediated RNA Decay (NMD)-pathway that is not only an RNA quality control mechanism but also has a role in regulating gene expression. PIWIL1 is a PIWI protein in the PIWI-interacting RNA (piRNA) pathway. PIWI proteins are mostly expressed in germ cells and are important in regulating the activity of piRNAs. They are important for transposon silencing and in meiotic and post-meiotic regulation of non-coding and protein-coding RNAs. A group of genes that seem to be regulated by both SMG6 and PIWIL1 has been identified with the help of high-throughput sequencing data (RNA sequencing) where the genes were upregulated in germ cells lacking both SMG6 and PIWIL1 (Prof. Noora Kotaja’s laboratory, University of Turku). The target genes of SMG6 and PIWIL1 that were to be characterized in my thesis were TATA-box binding protein associated factor RNA polymerase I D (Taf1d) and Cyclin Y (Ccny). The aim was to characterize the expression pattern of Taf1d and Ccny in mouse. This was done by performing immunofluorescent staining on mouse testes to find out in which cells they are expressed during spermatogenesis. RT-PCR on different mouse tissues was performed to conclude if they are testis-specific or ubiquitously expressed. Additional data analysis of RNA sequencing results from different time points during the first wave of spermatogenesis in wild-type mouse testis was done to identify the expression of Taf1d and Ccny during the first wave of spermatogenesis. In addition to the expression, the role of Taf1d in mouse spermatocyte GC-2spd cells was examined by a cell proliferation assay. The results show that Taf1d and Ccny mRNAs are ubiquitously expressed in different mouse tissues. Both RNA sequencing data and immunofluorescence analysis suggest that TAF1D and CCNY are widely expressed in different cells undergoing spermatogenesis.The silencing of Taf1d did not affect the proliferation of GC- 2spd cells. However, before concluding that TAF1D is not crucial for cell survival, several repeats are needed for the cell proliferation experiment.