Mss116p: a DEAD-box protein facilitates RNA folding

Abstract

RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA. DEAD-box proteins, including Mss116p, were recently found to assist folding of group I and group II introns in vitro and in vivo. The underlying mechanism(s) have been studied extensively to explore the contribution of ATP hydrolysis and duplex unwinding in helicase-stimulated intron splicing. Here we summarize the ongoing efforts to understand the novel role of DEAD-box proteins in RNA folding.

Keywords: DEAD-box proteins; RNA folding; RNA helicase; RNA-protein interactions; intron; ribozyme.

Figure 1. The DEAD-box helicase Mss116p and its conserved sequence motifs. Upper panel: Schematic representation of the helicase core region of the DEAD-box protein Mss116p. Mt, mitochondrial localization signal, which is cleaved off after import; NTE – N-terminal extension; CTE, C-terminal extension; C-tail, containing numerous basic amino acids. Functional regions shown in black or white, respectively, are not part of the 3D structure. Lower panel: Crystal structure of the Mss116p helicase core, which consists of two RecA-like domains, and its helical C-terminal extension. The conserved motifs are colored according to their primary function: red, ATP binding and hydrolysis; blue, RNA binding; yellow, communication between ATP binding and RNA binding sites. The non-conserved regions of the helicase domains are in gray and the CTE is shown in light-gray. The RNA is shown in pale yellow, the non-hydrolyzable ATP analog (AMPNP) in white and the Mg²⁺ ion in green. This figure has been adapted from references ,,. Note: in some recent helicase reviews on DEAD-box helicases, motif Ib has been renamed to motif Ic, while the GG doublet has become motif Ib, thus we also refer to the GG doublet as motif Ib and to the TPGRLID sequence as motif Ic as described in references ,,.

Figure 2. Interactions of the ATP analog and ssRNA with the helicase core. Left panel: Amino acids that were mutated to dissect Mss116p’s function in splicing of yeast mitochondrial introns are highlighted: K158, violet; S305, cyan; T307, dark cyan; Q412, blue; R245, red; I551, green; K569 – orange; the RNA is colored in pale yellow and AMPNP in white. The Mg²⁺ ion is represented as bright yellow sphere. Note that I551 and K569 are shown to indicate the start site of truncations in respective Mss116p mutants. Right panel: Conserved amino acids of the RecA-like domains contacting the non-hydrolyzable ATP analog (AMPNP; upper right panel) and RNA (lower right panel), respectively.

Figure 3. Mss116p-facilitated folding of the ai5γ intron in vitro. The DEAD-box protein accelerates compaction of intron domain D1 to a near-native folding intermediate, by stabilizing a specific structure at the core of this intron domain early in the ai5γ folding pathway. Subsequently, other intron domains can dock rapidly onto the D1 scaffold, whereby Mss116p does not stabilize the native state and is recycled upon ATP hydrolysis. D1 is shown in blue shades except for the κ−ζ element colored in purple shades, D3 is in green, D5 in red and D6 in yellow, while D2 and D4 are depicted in gray. Exons are outlined as thick black lines. Mss116p is indicated as orange ellipse. This figure has been adapted from references ,.