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

Cells (2020-04-23)
Maciej J Smialek, Erkut Ilaslan, Marcin P Sajek, Aleksandra Swiercz, Damian M Janecki, Kamila Kusz-Zamelczyk, Tomasz Wozniak, Maciej Kotecki, Luiza Handschuh, Marek Figlerowicz, Jadwiga Jaruzelska

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins (RBPs) with wide-ranging roles. They are involved in germ cell development, which has functional implications in development and 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. To address that problem, first we used RIP-Seq and RNA-Seq approaches, and identified mRNA pools regulated by PUM1 and PUM2 proteins in the TCam-2 cell line, a human male germ cell model. Second, applying global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-interacting putative protein cofactors, most of them involved in RNA processing. Third, combinatorial analysis of RIP and RNA-Seq, mass spectrometry, and RNA motif enrichment analysis revealed that PUM1 and PUM2 form partially varied RNP-regulatory networks (RNA regulons), which indicate different roles in human reproduction and testicular tumorigenesis. Altogether, this work proposes that protein paralogues with very similar and evolutionary highly conserved functional domains may play divergent roles in the cell by combining with different sets of protein cofactors. Our findings highlight the versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

Product Number
Product Description

JumpStart Taq DNA Polymerase, without MgCl2
Magna RIP® RNA-Binding Protein Immunoprecipitation Kit, RNA Immunoprecipitation (RIP) Kit containing all necessary reagents to perform 12 individual RNA-binding protein immunoprecipitation (RIP) reactions using protein A/G magnetic beads.
Anti-Actin antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, affinity isolated antibody
Actinomycin D, from Streptomyces sp., ~98% (HPLC)