Antagonistic and cooperative AGO2-PUM interactions in regulating mRNAs

Abstract

Approximately 1500 RNA-binding proteins (RBPs) profoundly impact mammalian cellular function by controlling distinct sets of transcripts, often using sequence-specific binding to 3′ untranslated regions (UTRs) to regulate mRNA stability and translation. Aside from their individual effects, higher-order combinatorial interactions between RBPs on specific mRNAs have been proposed to underpin the regulatory network. To assess the extent of such co-regulatory control, we took a global experimental approach followed by targeted validation to examine interactions between two well-characterized and highly conserved RBPs, Argonaute2 (AGO2) and Pumilio (PUM1 and PUM2). Transcriptome-wide changes in AGO2-mRNA binding upon PUM knockdown were quantified by CLIP-seq, and the presence of PUM binding on the same 3′UTR corresponded with cooperative and antagonistic effects on AGO2 occupancy. In addition, PUM binding sites that overlap with AGO2 showed differential, weakened binding profiles upon abrogation of AGO2 association, indicative of cooperative interactions. In luciferase reporter validation of candidate 3′UTR sites where AGO2 and PUM colocalized, three sites were identified to host antagonistic interactions, where PUM counteracts miRNA-guided repression. Interestingly, the binding sites for the two proteins are too far for potential antagonism due to steric hindrance, suggesting an alternate mechanism. Our data experimentally confirms the combinatorial regulatory model and indicates that the mostly repressive PUM proteins can change their behavior in a context-dependent manner. Overall, the approach underscores the importance of further elucidation of complex interactions between RBPs and their transcriptome-wide extent.

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

“…Although mammalian PUM1 and PUM2 contain nearly identical PUF domains [9] that recognize the same PBE motif (UGUANAUA) [10], there is some evidence for divergent modes of regulation. Examination of interactions between another RBP, ARGONAUTE2 (AGO2), and PUM proteins revealed a substantial fraction of nonoverlapping PUM1 and PUM2 mRNA targets [11]. Therefore, it is possible that PUM1 and PUM2 paralogues are functionally nonredundant and function as distinct RNA regulons.…”

MicroRNAs play an essential role in autophagy regulation in various disease phenotypes

“…Argonaute (Ago) protein is an RNA‐binding protein, which exists in the extracellular space as a free protein or in secreted exosomes along with other RNA‐processing proteins. Ago accumulates in cytoplasmic processing bodies (P‐bodies), where additional binding interactions promote mRNA decay and translational inhibition . A ribonucleoprotein complex called the RNA induced silencing complex (RISC), which is formed by Ago proteins, such as Ago‐2, and is involved in target recognition by small noncoding RNAs.…”

“…Ago accumulates in cytoplasmic processing bodies (P-bodies), where additional binding interactions promote mRNA decay and translational inhibition. [69][70][71] A ribonucleoprotein complex called the RNA induced silencing complex (RISC), which is formed by Ago proteins, such as Ago-2, and is involved in target recognition by small noncoding RNAs. Ago-2 protein separates mature miR duplexes into two strands, collectively known as the guide strand (miR) and the passenger strand (miR*).…”

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

“…The sequence preferences for both the full length PUM1 and PUM2 have been previously probed in vivo [36–38, 50] and the sequence preferences for the RNA-binding domains of both PUM1 and PUM2 were probed in vitro [10, 51, 52]. Each of these approaches and methodologies agree on a general preference for the UGUANAUA consensus motif for both PUM1 and PUM2, with subtle differences in the information content for the Position Weight Matrices (PWM)s obtained from each technique, particularly at the 3′ end of the PWM.…”