Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

Abstract

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

RNA remodeling and gene regulation by cold shock proteins

“…98 RNase II and RNase R belong to the RNR family. Based on sequence analysis and comparison with the RNase II structure, [99][100][101] RNase R consists of a central nuclease domain, two cold-shock (CSD) domains near the N-terminal region of the protein, an S1 domain and a highly basic C-terminal region. 102 While not detected in our complementation screen of csdA deletion strain (above) overproduction of RNase R or CspA complemented the cold-sensitive phenotype of the deletion strain.…”

Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

“…The catalytic activity is provided by a 10th essential subunit, Rrp44p (2,5,6). This protein is similar to RNase II in that it contains three putative RNA binding domains that flank an RNB domain (3,(7)(8)(9)(10). The RNB domain is responsible for the 3′ exonuclease activity of the exosome (3).…”

Poring over exosome structure

“…rrp44 contains an N terminal endonuclease domain-which seems to be relatively static-and a more flexible carboxy terminal part that resem bles bacterial rNase ii, as both contain three rNa binding domains-cSD1, cSD2 and S1-and a catalytic domain. three previous crystal structures of rNase ii and rrp44 (Frazao et al, 2006;Lorentzen et al, 2008;Bonneau et al, 2009) had different orien tations of cSD1 and cSD2 relative to the rest of the protein. Malet et al (2010) found that cSD1 is slightly shifted compared with previous structures and that cSD2 does not display well ordered density in the absence of rNa, but becomes more ordered in its presence (Malet et al, 2010).…”