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.
Highlights
Post-transcriptional gene regulation (PTGR) is crucial for maintaining cellular proteome homeostasis [1,2], disruption of which can cause severe diseases, such as cancer and infertility [3].PTGR requires the activity of RNA-binding proteins (RBPs), such as the widely studied pumilio (PUM) proteins, which are founding members of the PUF familyCells 2020, 9, 984; doi:10.3390/cells9040984 www.mdpi.com/journal/cellsCells 2020, 9, 984 of eukaryotic RBPs
We found that PUM-binding element (PBE) were mostly located in the 30 untranslated regions (30 UTR) (88% and 83% for PUM1 and PUM2, respectively), less frequently in CDS (10% and 15% for PUM1 and PUM2, respectively), and rarely in the 50 UTR
53 activated) PUM2-regulated (Figure 1H lower panel, Table S10) mRNAs. By using these two criteria, we found that the number of mRNAs shared by PUM1 and PUM2 was reduced to 10% (47 common mRNAs) (Figure 1I) compared to the 30% seen by RNA immunoprecipitation (RIP)-Seq-based selection (Figure 1A)
Summary
PTGR requires the activity of RNA-binding proteins (RBPs), such as the widely studied pumilio (PUM) proteins, which are founding members of the PUF (pumilio and fem-3 binding factor) family. Cells 2020, 9, 984 of eukaryotic RBPs. PUM proteins are highly conserved and present in many organisms, from yeast to humans (for review, see [4]). Post-transcriptional regulation by PUMs is mediated by the conserved C-terminal RNA-binding PUF domain, which is composed of eight tandem repeats [6], and binds a specific eight nucleotide sequence 50 -UGUAHAUA-30 (H represents A, C or U, but not G), called the PUM-binding element (PBE) that is typically located in the 30 untranslated regions (30 UTR). Of target mRNAs. By binding PBEs, PUMs trigger the recruitment of protein cofactors, that together direct selected mRNAs towards post-transcriptional repression or activation (for review see [4])
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