Inputs (0.5%) and anti-FLAG-IPs (20%) were analyzed by SDS-PAGE and western blot using antibodies against DHX34, RUVBL1 and RUVBL2.
( E) IP experiment testing the interaction of FLAG-RUVBL1 and HA-RUVBL2 co-expressed in HEK293T cells with T7-DHX34. For Inputs (0.5%) and anti-T7 IP (20%) are shown. Proteins bound to T7 tag affinity beads were eluted and analyzed by SDS-PAGE and western blot using antibodies against the T7 tag in DHX34 and RUVBL1 ( C) or RUVBL2 ( D). Inputs (0.5%) and anti-FLAG-IPs (20%) were subjected to western analysis using the indicated antibodies. ( C, D) Immunoprecipitation (IP) of transiently transfected HEK293T cells with T7-DHX34 from HEK293T cells was performed in the presence of RNase A. DHX34 was found to elute specifically only when His-RUVBL1-RUVBL2 was present. Proteins bound to affinity beads were eluted and analyzed by SDS-PAGE and stained using Oriole Fluorescent Gel Stain (Bio-Rad). ( B) Pull-down experiment testing the interaction of purified His-RUVBL1-RUVBL2 with DHX34, using His-tag affinity purification.
The ATPase-face and DII-face of the ring as well as the internal and external regions of DIIs are indicated.
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Top and bottom views are shown with the color code from top panel. ( A) Top panels: Schematic representation of RUVBL1 (blue) and RUVBL2 (pink) domains and catalytic motifs. Bottom panel: structure of the human RUVBL1-RUVBL2 hetero-hexameric ring with protruding domain II (DII), generated from atomic structures of RUVBL1 (PDB 2C9O) and RUVBL2 (PDB 6H7X) ( Matias et al., 2006 Silva et al., 2018). Several structures of RUVBL1-RUVBL2 complexes reveal that nucleotides are present in the complex even if they were not supplemented during purification ( Gorynia et al., 2011 Lakomek et al., 2015 Matias et al., 2006 Muñoz-Hernández et al., 2019). Each RUVBL1 and RUVBL2 subunit contains a nucleotide-binding pocket and the adjacent subunit in the hexamer provides the arginine finger motif required for hydrolysis. DII domains comprise an oligonucleotide/oligosaccharide-binding (OB) fold domain (DII external) that connects to the hexameric ring by a flexible region containing a ß-stalk and a helical bundle (DII internal). RUVBL1 and RUVBL2 contain a unique domain II (DII) that protrudes from one side of the hexamer and defines two distinct faces of the ring, named ATPase-face and DII-face hereafter ( Cheung et al., 2010 Ewens et al., 2016 Gorynia et al., 2011 Lakomek et al., 2015 López-Perrote et al., 2012). RUVBL1 and RUVBL2 are two closely related AAA-type ATPases that assemble as hetero-hexameric structures made of alternating subunits and comprising six ADP/ATP-binding domains ( Figure 1A). We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. 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. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. 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.
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