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.
Highlights
Post-transcriptional control of gene expression is central to a wide range of cellular processes, ensuring proper cell homeostasis
To further investigate potential AGO2-PUM co-regulatory interactions, crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) experiments were performed for the endogenous PUM1, PUM2 and AGO2 proteins in 293 cells and derivatives (Supplementary File 1)
The greater number of transcripts may be explained by a higher binding affinity, or additional specificity determinants associated with PUM2 binding
Summary
Post-transcriptional control of gene expression is central to a wide range of cellular processes, ensuring proper cell homeostasis. The regulation is mainly accomplished by RNA-binding proteins (RBPs) and microRNAs (miRNAs), both of which target mature messenger RNAs by binding to defined sites in the 3′ untranslated region (UTR) Often, these specific factors serve as the target recognition components of larger complexes, which recruit additional, nonspecific factors to stabilize or repress target mRNA expression. Pumilio proteins have been documented to have stabilizing effects on target mRNAs across eukaryotes[34,44,48,49,50,51] The mechanisms underlying these biologically relevant effects appear to be case-specific and may involve interaction with additional factors and/or changes in 3′UTR secondary structure upon Pumilio binding
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