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

Abstract

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.

Role of PUM RNA-Binding Proteins in Cancer

“…In total, 346 PUM1-regulated and 141 PUM2-regulated targets were identified in that study. About 90% of PUM-regulated targets were different for PUM1 and PUM2, and nearly 100% of all identified targets contained PBEs, thus validating the results [ 34 ]. Several other studies aimed at the global identification of human PUM1 and PUM2-regulated targets were performed in other human cell lines and using some methodological modifications compared to the study in TCam-2 cells.…”

“…A global immunoprecipitation (IP) and mass spectrometry (MS)-based identification of PUM1- and PUM2-binding proteins showed that PUM1 and PUM2 indeed interact mainly with different groups of proteins, a majority of them representing RBPs. Combinatorial analysis of RNA immunoprecipitation (RIP) and RNA-Seq MS, as well as motif enrichment analysis for RNA-binding proteins in PUM1 and PUM2 mRNA targets, revealed that PUM1 and PUM2 form separate ribonucleoprotein networks [ 34 ], which resemble so-called “regulons” or “posttranscriptional operons”, as previously suggested for RBPs that recognize specific motifs in RNAs [ 41 ]. According to those networks, PUM1 and PUM2 may cooperate with varied protein cofactors to regulate mRNA target sub-pools responsible for unique pathways.…”

“…For example, PUM1 with IGF2BP3 cofactor may co-regulate mRNAs implicated in regulation of cell division, while PUM1 with PABPC4 and MATR3 may co-regulate mRNAs involved in the epidermal growth factor receptor-signaling pathway. On the other hand, PUM2 with G3BP2, HNRNPA1, FXR2, and SFPQ co-factors may co-regulate mRNAs involved in the regulation of Rho protein signal transduction, while with MATR3, PUM2 may affect mRNAs that negatively regulate cell development [ 34 ]; however, such scenarios require validation at the protein level. One important aspect that should be taken into account when discussing the functional relationship between PUM1 and PUM2 interactomes is that PUM1 and PUM2 mRNAs contain functional PBEs in their 3′UTRs.…”

Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

“…We did not include NANOS2 in this study because it is not expressed at that early stage of germline development represented by the TCam-2 cells [ 16 ]. Our transcriptome analysis of this cell line by RNA-sequencing (RNA-Seq) showed that both NANOS1 and NANOS3 are expressed at low levels at that stage (1.7 and 2.1 FPKM, respectively) [ 29 ]. Therefore, we overexpressed NANOS1 and NANOS3, followed by RNA sequencing (RNA-Seq) and differential gene expression (DGE) analysis to identify mRNAs potentially downregulated by these post-transcriptional repressors.…”

“…We have previously identified FOXM1 mRNA as a direct target of PUM1 [ 29 ]. Thus, we asked whether NANOS3 and PUM1 cooperate in FOXM1 mRNA repression.…”