A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation

Abstract

Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.

Structural basis for clearing of ribosome collisions by the RQT complex

“…In the first step, the RQT is recruited to the ribosome queue after Hel2-dependent K63-linked uS10 polyubiquitination via interaction with the CUE domain of the Cue3 subunit 12 , 22 , 46 , 47 . After recruitment, RQT stably locks onto the 40S of the stalled/lead ribosome in the C1 conformation via the Slh1 NTC, placing the Slh1 CTC in a position primed to bind the 3′ region of the mRNA emerging from the ribosomal mRNA entry site.…”

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex

“…A recent study demonstrated that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation, 16 indicating that the crucial role of K63-linked ubiquitination of uS10 for the RQT-mediated clearance is conserved in eukaryotes from yeast to mammals. Since the trimeric hRQT complex is composed of ASCC3 (Slh1), ASCC2 (Cue3), and TRIP4 (Rqt4), the conservation of the mechanism for K63-linked ubiquitin recognition by TRIP4 should be investigated in the future work.…”

“…Persistent translation arrest results in ribosome collision, which is considered a hallmark of translational stress. [1][2][3][4][5][6][7] Several sensor proteins recognize ribosomal collision and induce different cellular responses, including the ribosome-associated quality control (RQC) pathway, [8][9][10][11][12][13][14][15][16] no-go decay (NGD), [17][18][19] the integrated stress response (ISR), [20][21][22][23][24][25] and the ribotoxic stress response (RSR). 21,[26][27][28] The RQC pathway is the rescue system for stalled ribosomes, thereby contributing to the clearance of collided ribosomes to decrease the severity of translational stress.…”

Mechanistic insights into the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control

“…Anisomycin treatment increases the ZNF598-mediated ubiquitination of uS10 and eS10 and their disome formation (50)(51)(52)(53) and also increases ufmylation, suggesting that ufmylation is required for ER-RQC. We examined the role of the UFL1-UFBP1-CDK5RAP3 E3 complex in RQC-mediated degradation of nascent polypeptides derived from stalled ribosomes.…”