Kinesin KIF18A is a novel PUM regulated target promoting mitotic progression and survival of human male germ cell line - TCam-2

Abstract

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. Kinesin KIF18A is an important germ cell specific regulator which downregulates apoptosis while promoting cell proliferation in animal models. Whereas regulation of KIF18A expression was studied at the transcriptional level, its posttranscriptional regulation has not been extensively explored. Due to the presence of two PUM Binding Elements (PBEs) within 3UTR, KIF18A mRNA is a potential target of PUMs, well known RNA-binding proteins involved in posttranscriptional gene regulation (PTGR). We investigated that possibility in TCam-2 cells originating from seminoma, representing human male germ cells. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assay by applying appropriate luciferase construct encoding the wild type KIF18A 3UTR, upon PUM1 and PUM2 overexpression or knockdown. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function in TCam-2 cells, MTS proliferation assay, apoptosis and cell cycle, analysis using flow cytometry was performed upon KIF18A siRNA knockdown. We uncovered that KIF18A significantly influences proliferation, apoptosis and cell cycle, these effects being opposite to PUM effects in TCam-2 cells. We propose that repression by PUM proteins may represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells. To the best of our knowledge, this paper identifies the first mammalian functionally germ cell specific gene that is regulated by Pum proteins via 3UTR.