The structure of Prp2 bound to RNA and ADP-BeF₃⁻reveals structural features important for RNA unwinding by DEAH-box ATPases

Abstract

Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3 − and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3 −- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3′ stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

Functional and Structural Characterization of the DEAH-box Helicase Prp43

“…In absence of Pfa1(gp), both Prp43(wt) and Prp43Cys unwind the substrate extremely slowly (kobs=0.003 s-1, Figure 2C), while the addition of Pfa1(gp) leads to comparably faster unwinding (kobs=0.36 sand kobs=0.34 s -). This indicates a strong stimulation of Prp43 helicase activity by Pfa1(gp) and is in line with previous measurements on ctPrp43 (Tauchert et al, 2017;Hamann et al, 2021). In summary, the central determinants of Prp43 function, i.e.…”

“…In Prp2, which does not display in vitro unwinding activity, this C-loop interacts with a loop in the RecA2 domain instead. As the sequence of the C-loop differs between the four spliceosomal DEAH-box helicases but is conserved in each individual helicase among different organisms, it likely plays a role in regulating their unwinding activity (Hamann et al, 2021).…”

Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1

“…Further domains include a common set of C-terminal domains composed of a winged helix (WH), a helix bundle (HB, previously called ratchet-like domain), and an oligosaccharide binding (OB) -fold domain as well as varying N-terminal extensions ( 5 , 6 ). RNA is bound in a sequence-independent manner in a channel between the RecA and the C-terminal domains ( 7 – 12 ). A mechanistic model for single strand RNA (ssRNA) translocation has been developed from crystal structures of DEAH/RHA helicases in different conformational states ( 7 – 10 , 13 , 14 ): Transitions between open, nucleotide-free and closed, nucleotide-bound conformations of the helicase core lead to alternate accommodation of either four or five RNA nucleotides stacked in the binding channel with a conserved loop in RecA1 and a β-hairpin in RecA2 serving as “bookends” preventing slippage.…”

Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions

“…6A– C) [61]. The hydrolysis of ATP enables the release of ADP and phosphate, while the phosphate ion is predominantly released via a tunnel on the backside of Prp43 [25]. Removal of these negative charges leads to a springlike conformational change in the ATP binding pocket, driving the movement of RecA2 by one RNA base upstream (Fig.…”