The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

Abstract

1Cells have evolved quality control mechanisms to ensure protein homeostasis by 2 detecting and degrading aberrant mRNAs and proteins. A common source of aberrant 3 mRNAs is premature polyadenylation, which can result in non-functional protein 4 products. Translating ribosomes encountering poly(A) sequences are terminally 5 stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide 6 via the ribosome-associated quality control (RQC). Here, we demonstrate that the 7 conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) 8 promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 9 is positioned upstream of A-rich stretches and poly(A) tails in mRNAs through an 10 interaction with the cytoplasmic poly(A)-binding protein (PABP). We uncover PABP, 11 ribosomal protein RPS10, and additional translational regulators as main ubiquitylation 12 substrates of MKRN1. Consequently, we propose that MKRN1 serves as a first line of 13 poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus 14 maintaining proteome integrity. 15 Keywords 16 MKRN1, ubiquitylation, RNA binding, ribosome-associated quality control, RQC, 17 poly(A), iCLIP, ubiquitin remnant profiling, translation 18 PABP-interacting motif 2 (PAM2 motif) in rat neurons (Miroci et al. 2012). 61 Nevertheless, the RNA binding specificity and functional role of MKRN1 in human cells 62 remained largely elusive. 63Here, we introduce MKRN1 as a novel factor in RQC. MKRN1 is recruited to A-rich 64 sequences in mRNAs in a PABP-dependent manner, where it acts as a first line of 65 defence against poly(A) translation. MKRN1 depletion abrogates ribosome stalling in 66 reporter assays, accompanied by reduced ubiquitylation of RQC-related proteins. We 67

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development

“…For instance, the AKT2 gene product exhibited an increased accumulation of MKRN2 iCLIP reads within the 3 0 UTR relative to other transcript regions (Figure 5E). Hence, our iCLIP analysis indicates that MKRN2 binds to mRNAs primarily in the 3 0 UTR, consistent with recent iCLIP analyses of another Makorin family member, MKRN1, in human cells (Hildebrandt et al, 2019) and Drosophila (Dold et al, 2020).…”

“…Previous AP-MS analyses of smaller subsets of mammalian E3-RBPs have identified interactions with other RBPs and RNA-associated protein complexes (Cano et al, 2015;Cassar et al, 2015;Hildebrandt et al, 2017Hildebrandt et al, , 2019Leppek et al, 2013;Katzenellenbogen et al, 2007;Yellapragada et al, 2019). Consistent with a preponderance of E3-RBP functions in RNA processing and related pathways, annotated RNA-binding proteins were significantly overrepresented (p value = 1.17 3 10 À58 by hypergeometric testing) in the set of high-confidence interacting ''prey'' proteins that we identified (Figure S1A).…”

“…GraphProt (Maticzka et al, 2014) analysis of the sequences surrounding mapped MKRN2 cross-linking sites, relative to a control background of random sequences from protein-coding genes expressed in HEK293 cells (see STAR Methods), revealed enrichment of a CU-rich motif in predominantly structured regions (Figure 5F) and distributed slightly upstream of cross-linking sites (Figure S4G). Notably, this is distinct from the reported A-rich recognition motif of human MKRN1 (Hildebrandt et al, 2019), possibly due to functional divergence between MKRN1 and MKRN2.…”

“…Next, we specifically examined the interactions of the Makorin family of E3-RBPs. Previous studies have identified physical binding between many Makorin family members and poly(A) binding proteins (PABPs) (Cassar et al, 2015;Dold et al, 2020;Hildebrandt et al, 2017Hildebrandt et al, , 2019Miroci et al, 2012;Yellapragada et al, 2019), which are involved in diverse RNA-associated activities (Mangus et al, 2003). Consistent with this previous work, we similarly observed high-confidence interactions between all human Makorin family members and PABPs (Figure S1B).…”

“…MKRN2 is one of at least 26 putative human E3-RBPs that have been shown or predicted to bind RNA in human cells (Hildebrandt et al, 2017). While some have been linked to RNA processing, such as RC3H1 in mRNA turnover (Leppek et al, 2013) and MKRN1 in the regulation of translation (Dold et al, 2020;Hildebrandt et al, 2019;Miroci et al, 2012), many E3-RBPs lack known RNA-associated interaction partners or functions in RNA processing.…”