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

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. The kinesin KIF18A is an important regulator of those processes in animal germ cells. Post-transcriptional regulation of KIF18A has not been extensively explored. Owing to the presence of PUM-binding elements (PBEs), KIF18A mRNA is a potential target of PUM proteins, where PUM refers to Pumilio proteins, RNAbinding proteins that act in post-transcriptional gene regulation. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assays, by applying an appropriate luciferase construct encoding wild-type KIF18A 3′-UTR, upon PUM overexpression or knockdown in TCam-2 cells, representing human male germ cells. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function, an MTS proliferation assay, and apoptosis and cell cycle analysis using flow cytometry, was performed upon KIF18A mRNA siRNA knockdown. KIF18A significantly influences proliferation, apoptosis and the cell cycle, with its effects being opposite to PUM effects. Repression by PUM proteins might represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells.

Characterization of RNP Networks of PUM1 and PUM2 Post-Transcriptional Regulators in TCam-2 Cells, a Human Male Germ Cell Model

“…This may reflect a mutual regulation of PUM proteins, as it was previously reported that PUM1 represses PUM2 mRNA and vice versa, since they both contain PBEs in 3 UTR [53]. We have observed this phenomenon of mutual PUM1 and PUM2 repression in TCam-2 cells [54]. Hence, we propose that knockdown of one PUM paralogue activates a feedback, resulting in upregulation of the other PUM.…”

Kinesins in Mammalian Spermatogenesis and Germ Cell Transport

“…Cell lines or cultured primary cells obtained from humans could be a viable alternative to animal models, providing mechanistic insights without demanding fresh primary cultures from human testes. TCam-2, a human tumor cell line derived from a primary testicular seminoma of a 35-year-old men and assumed to reflect human male germ cells, was utilized to investigate KIF18A activity in proliferation, cell cycle and apoptosis in the human cell line ( Smialek et al, 2020 ). The use of a well-established human Sertoli cell in vitro system sourced from cadaveric testes of males aged 12–36 years could be a useful tool for studying the role of kinesins in human Sertoli cell function and BTB dynamics ( Chui et al, 2011 ; Xiao et al, 2014b ; Chen et al, 2017 ).…”

Role of PUM RNA-Binding Proteins in Cancer

“…In TCam-2 cells, PUMs also cause repression of mRNA encoding kinesin KIF18A [ 49 ]. This kinesin exists on positively charged ends of microtubules in the vicinity of the kinetochore, and it regulates the dynamics of mitotic cell division.…”

“…It has been reported that KIF18A positively influences TCam-2 cell proliferation, downregulates apoptosis, and promotes cell cycle progression, these effects being opposite to the effects of PUMs. Therefore, repression by PUM proteins may represent one of the mechanisms affecting KIF18A levels in regulating proliferation, the cell cycle, and apoptosis in TCam-2 cells [ 49 ].…”