Intrinsic Dynamics of an Extended Hydrophobic Core in the S. cerevisiae RNase III dsRBD Contributes to Recognition of Specific RNA Binding Sites

Abstract

The S. cerevisiae RNase III enzyme Rnt1p preferentially binds to dsRNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its dsRBD helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin relaxation experiments. The intrinsic μs-ms timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1-β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and non-specific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity, and cause defects in snoRNA processing in vivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.

Ribonuclease III mechanisms of double‐stranded RNA cleavage

“…Rnt1p recognizes the tetraloop in a shape‐dependent (rather than sequence‐dependent) manner, and cleaves the stem ∼14–16 bp from the structure. Tetraloop recognition involves the dsRBD α 1 helix, which possesses an extended structure relative to the α 1 helix of other RNases III . The elongated helix provides an expanded hydrophobic core, conferring a conformational plasticity to the dsRBD that is important for forming a functionally competent dsRBD‐hairpin complex .…”

Post-Transcriptional Regulation of Iron Homeostasis in Saccharomyces cerevisiae

“…The conformational flexibility of Rnt1 dsRBD allows the specific recognition of the double-stranded RNA hairpin tetraloop structures capped by a conserved (A/u)GNN motif, whereas its RNase III domain triggers mRNA cleavage to generate 5′-monophosphate and 3′ hydroxyl termini with a two-base 3′ overhang [102–107]. Rnt1 cleavage products are usually further processed in the nucleus by the Rat1 5′ exonuclease and the 3′ exosome.…”

Structure of a Eukaryotic RNase III Postcleavage Complex Reveals a Double-Ruler Mechanism for Substrate Selection

“…However, it is not clear how Rnt1p selects these substrates and how the enzyme positions its cleavage site at a fixed distance from the tetraloop. Until now, the exact contribution of the tetraloop, especially the conserved guanine nucleotide, to substrate binding and cleavage is still being debated (Ghazal and Elela, 2006; Hartman et al, 2013; Lavoie and Abou Elela, 2008; Wang et al, 2011; Wu et al, 2004). …”