Vault RNA1–1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization

Abstract

Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional and post-translational mechanisms. We have recently shown that the small, non-coding vault RNA1-1 negatively riboregulates p62 oligomerisation in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerisation are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.

Small but Powerful: The Human Vault RNAs as Multifaceted Modulators of Pro-Survival Characteristics and Tumorigenesis

“…Mutational analysis revealed that the disruption of this short domain on vtRNA1-1, similarly to the vault RNA KO, increases apoptosis. This short stretch is located in the central region of the RNA, where the secondary structure features a single-stranded loop region and a stem-loop [ 48 , 58 ]. This domain has been shown to bind to p62 (sequestosome1 protein SQSTM1) [ 58 ] and is possibly also the anchor point for other vtRNA1-1 interacting proteins, although the binding partners of the vault RNA orchestrating apoptosis have not been uncovered yet.…”

“…This short stretch is located in the central region of the RNA, where the secondary structure features a single-stranded loop region and a stem-loop [ 48 , 58 ]. This domain has been shown to bind to p62 (sequestosome1 protein SQSTM1) [ 58 ] and is possibly also the anchor point for other vtRNA1-1 interacting proteins, although the binding partners of the vault RNA orchestrating apoptosis have not been uncovered yet. An initial mechanistic hint was recently published, suggesting a possible direct interaction of mouse vtRNA with MEK1, thus stimulating MAPK signaling and synaptogenesis [ 59 ].…”

“…This ensures the biogenesis of catabolically active lysosomes (acidic pH) capable of fusing with autophagosomes to form active autolysosomes [ 49 ]. Moreover, vaultRNA1-1 is able to inhibit p62 oligomerization and has been suggested to regulate the available cargo of ubiquitinated proteins for the autophagosome (green T-bar) [ 58 , 64 ]. The relevant residues on vtRNA1-1 for this proposed role are encircled in green.…”

Crosstalk between vault RNAs and innate immunity

“…Mutational assays targeting this RNA segment have shown an increase in apoptosis in HeLa cells treated with the drug chloroquine and subjected to nutrient deprivation using starvation medium [ 128 ]. Moreover, this domain has been shown to interact with p62, a protein involved in autophagy [ 138 ]. As a result, it may also serve as a central point for interactions with other RBPs (Fig.…”

“…The stem-loop structure in the apical region of vtRNA 1-1 enhances resistance to apoptosis by modulating the PI3K/Akt and ERK1/2 MAPK signaling pathways, with critical nucleotides for apoptosis resistance encircled in blue [ 87 , 127 ]. Additionally, vtRNA 1-1 inhibits autophagy by preventing p62 oligomerization, and the relevant nucleotides for this proposed role are encircled in red [ 138 , 143 ]. Moreover, vault particles can interact with SQSTM1/p62, contributing to the enhancement of the vault-phagy process [ 141 ] …”