Biology subjects

Smialek, M. J.

Publications and source records attributed to Smialek, M. J..

3 recordsLinked to original sources

bioRxiv2019

A male infertility mutation reverts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes

BackgroundWhile Nanos-mediated maintenance of germ cells in Drosophila and mice has been related to regulation of apoptosis, the relevance of these findings to human physiology is uncertain. Previously we have described the p.[(Pro34Thr);(Ser83del)] double NANOS1 mutation as associated with an absence of germ cells in the testes of infertile patients. The aim of this study was to identify the mechanism underlying infertility phenotype of patients bearing the NANOS1 mutation. MethodsConstructs encoding a wild-type or mutated NANOS1 protein were used for transfection of TCam-2 cell line, representing male germ cells in culture. Influence of this mutation on cell proliferation was performed using MTS assay while apoptosis and cell cycle were measured by flow cytometry. RNA-Seq analysis including quantitative RT-PCR was conducted for selecting pro-apoptotic genes, repressed by the wild-type NANOS1. Influence of the p.[(Pro34Thr);(Ser83del)] NANOS1 mutation on that repression was investigated by quantitative RT-PCR. ResultsWe show here that the p.[(Pro34Thr);(Ser83del)] double NANOS1 mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line TCam-2. This mutation disrupts repression of mRNAs encoding pro-apoptotic GADG45A, GADD45B, GADD45G and RHOB factors, which could contribute to an increase in apoptosis. ConclusionsThis report underscores the conservation of Nanos from flies to humans as a repressor of pro-apoptotic mRNAs in germ cells, and provides a basis for understanding NANOS1 functions in human reproductive health.

developmental biology↗

bioRxiv2019

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

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.

developmental biology↗

bioRxiv2019

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins with wide-ranging roles, including germ cell development that has functional implications in fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity, particularly related to their roles in fertility. Here, by RNA sequencing (RNA-Seq) approaches, we identified separate mRNA pools regulated by PUM1 and PUM2 proteins in human male germ cells. Using global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-bound putative protein cofactors, most of them involved in RNA processing. Combinatorial analysis of RNA-Seq and mass spectrometry findings revealed that PUM1 and PUM2 may form distinct RNA-regulatory networks, with different roles in human reproduction and testicular tumorigenesis. Our findings highlight the functional divergence and versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

molecular biology↗