Molecular Mechanisms for the RNA-Dependent ATPase Activity of Upf1 and Its Regulation by Upf2

Abstract

Upf1 is a crucial factor in nonsense-mediated mRNA decay, the eukaryotic surveillance pathway that degrades mRNAs containing premature stop codons. The essential RNA-dependent ATPase activity of Upf1 is triggered by the formation of the surveillance complex with Upf2-Upf3. We report crystal structures of Upf1 in the presence and absence of the CH domain, captured in the transition state with ADP:AlF₄⁻ and RNA. In isolation, Upf1 clamps onto the RNA, enclosing it in a channel formed by both the catalytic and regulatory domains. Upon binding to Upf2, the regulatory CH domain of Upf1 undergoes a large conformational change, causing the catalytic helicase domain to bind RNA less extensively and triggering its helicase activity. Formation of the surveillance complex thus modifies the RNA binding properties and the catalytic activity of Upf1, causing it to switch from an RNA-clamping mode to an RNA-unwinding mode.

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

“…Similarly to what has previously been observed in the structures of nucleotide‐bound Upf1 Hel (Chakrabarti et al , 2011), the adenine ring is sandwiched between an apolar surface of RecA1 and an aromatic residue (Tyr1655) that is present in the linker connecting RecA1 to RecA2 and is part of motif IIIa (Fairman‐Williams & Jankowsky, 2012). In addition, the conserved side chain of Gln1339 forms a bidentate hydrogen‐bond interaction with the N6 and N7 moieties of the adenine ring (Fig EV2A).…”

“…The structure was refined at 1.8 Å resolution with R free of 18%, R factor of 15%, and good stereochemistry (Table 1) (Fig EV1C). Overall, Sen1 Hel has a domain organization similar to that of the helicase core of Upf1 (Upf1 Hel , also known as Upf1‐ΔCH) (Cheng et al , 2007; Clerici et al , 2009; Chakrabarti et al , 2011) as well as IGHMBP2 (IGHMBP2 Hel ) (Lim et al , 2012) (Fig 2). In the Sen1 Hel ‐ADP structure, the two RecA domains are positioned side by side, separated by a cleft about 10 Å wide (Fig 2).…”

“…Comparison of the structures of yeast Sen1 Hel ‐ADP, human UPF1 Hel ‐AMPPNPP (PDB: 2GJK, Cheng et al , 2007), UPF1 Hel ‐ADP:AlF 4 − ‐RNA (PDB: 2XZO, Chakrabarti et al , 2011), and yeast Upf1 Hel‐CH ‐ADP:AlF 4 − ‐RNA (PDB: 2XZL, Chakrabarti et al , 2011). The molecules in a side‐view orientation (90° clockwise rotation around a vertical axis with respect to the front‐view in Fig 4A).…”

“…Comparison of the RNA‐binding sites of Sen1 (left) and Upf1 (right) (PDB: 2XZO, Chakrabarti et al , 2011). …”

“…SF1B RNA helicases contain two RecA domains (RecA1 and RecA2) with the classical helicase motifs involved in nucleic acid binding and ATP hydrolysis. Helicases of this family also contain two SF1B‐specific subdomains (1B and 1C) that modulate RNA binding (Cheng et al , 2007; Clerici et al , 2009; Chakrabarti et al , 2011; Lim et al , 2012). SF1B helicases bind nucleic acids with the same polarity as all other RNA‐dependent ATPases, that is, with the 3′ end at RecA1 and the 5′ end at RecA2 (reviewed in Pyle, 2008; Ozgur et al , 2015).…”