Prolyl 4-hydroxylation regulates Argonaute 2 stability

Abstract

Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference 1 . Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies 1,2 . However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the α-(P4H-α(I)) and β-(P4H-β) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-α(I) or P4H-β by short hairpin RNA and P4H-α(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.To identify the protein network involved in regulating the RNA interference (RNAi) machinery, we established stable HeLa S3 cell lines expressing Flag-HA(haemagglutinin)-tagged human Ago1−4, respectively. Cytoplasmic extracts were immunoprecipitated with sequential anti-Flag and anti-HA antibody resins. Silver staining of a representative purification (Ago2) is shown in Fig. 1a. Mass spectrometric analysis of the purifications

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

“…To date, a limited number of studies have addressed the functional role of PTMs of miRNA pathway machinery. In particular, several PTMs to Argonaute have been characterized (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). However, little is known about the identity and functional significance PTMs in other components of miRISC.…”

hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

“…It may also reflect post-translational processes such as prolyl 4-hydroxylation: the same process that regulates von Hippel-Lindau gene, and in turn HIFm, regulates Argonaute 2 stability. 40 There was a good correlation between the expression of the pre-microRNAs containing hsa-miR-210 and expression by real-time PCR for the mature sequence. Mining of existing arrays is a much less labor-intensive process than real-time PCR for individual microRNAs, but only limited arrays can be mined, as not all microarrays have the relevant probe.…”

The complexity of miRNA-mediated repression

“…In addition to the above-mentioned phosphorylation events, Ago2 hydroxylation has been shown to stabilise the protein and enhance miRISC function following hypoxia. 119,120 Conversely, ADP-ribosylation of Ago2 upon oxidative stress has been shown to lead to the relief of miRNAmediated repression. 121 It is possible that different stress stimuli result in different responses of the miRNA machinery.…”