Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM.

Andres López-Perrote,Ana González-Corpas,Rafael Fernandez-Leiro,Nele Hug,Javier F Caceres,Carmen García-Martín,Marina Serna,Oscar Llorca,Carlos F Rodríguez,Jasminka Boskovic

doi:10.7554/elife.63042

Andres López-Perrote, Ana González-Corpas + Show 8 more

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https://doi.org/10.7554/elife.63042

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Abstract

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

Highlights

RUVBL1 and RUVBL2 are two closely related AAA-type ATPases that assemble as heterohexameric structures made of alternating subunits and comprising six ADP/ATP-binding domains (Figure 1A)
To uncover the function of RUVBL1 and RUVBL2 in Nonsense-mediated mRNA decay (NMD), we sought to address whether these AAA-ATPases can interact directly with some of the core protein factors involved in the initiation of NMD
RUVBL1 and RUVBL2 were co-expressed and purified as heteromeric complexes containing His-RUVBL1 and RUVBL2 in equimolar amounts, and forming oligomeric complexes where two hexameric rings interact though the domain II (DII) domains, as revealed by Cryo-electron microscopy (cryo-electron microscopy (EM)) (Figure 1 — figure supplement 1A), and in agreement with what we described before (Lopez-Perrote et al, 2012; Martino et al, 2018)

Summary

Introduction

RUVBL1 and RUVBL2 are two closely related AAA-type ATPases that assemble as heterohexameric structures made of alternating subunits and comprising six ADP/ATP-binding domains (Figure 1A). RUVBL1 and RUVBL2 interact with RPAP3 and PIH1D1 proteins to form the R2TP complex, a HSP90 co-chaperone involved in the assembly and maturation of some large complexes including RNA polymerase II and members of the Phosphatidylinositol 3-kinase-related kinase (PIKK) family such as ATR, ATM, SMG1 and mTOR (Houry et al, 2018; Martino et al, 2018; Maurizy et al, 2018; Munoz-Hernandez et al, 2019; Rivera-Calzada et al, 2017). One exception is the C-terminal domain of RPAP3 that binds at the ATPase-face of the ring through the interaction with RUVBL2 but not RUVBL1 (Martino et al, 2018; Maurizy et al, 2018; Munoz-Hernandez et al, 2019)

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