Structure of the Active Subunit of the Yeast Exosome Core, Rrp44: Diverse Modes of Substrate Recruitment in the RNase II Nuclease Family

Abstract

The eukaryotic exosome is a macromolecular complex essential for RNA processing and decay. It has recently been shown that the RNase activity of the yeast exosome core can be mapped to a single subunit, Rrp44, which processively degrades single-stranded RNAs as well as RNAs containing secondary structures. Here we present the 2.3 A resolution crystal structure of S. cerevisiae Rrp44 in complex with single-stranded RNA. Although Rrp44 has a linear domain organization similar to bacterial RNase II, in three dimensions the domains have a different arrangement. The three domains of the classical nucleic-acid-binding OB fold are positioned on the catalytic domain such that the RNA-binding path observed in RNase II is occluded. Instead, RNA is threaded to the catalytic site via an alternative route suggesting a mechanism for RNA-duplex unwinding. The structure provides a molecular rationale for the observed biochemical properties of the RNase R family of nucleases.

CryoEM structure of yeast cytoplasmic exosome complex

“…Compared with the RNA-free complex, the RNA-bound complex had its Rrp44-CSD1 domain moving away from the Rrp44-RNB domain, enlarging the RNA entry channel of Rrp44 so that its RNase active site is more accessible (right panels in Figure 4A, 4B and Supplementary information, Movie S4). There is also a loop (residues 705-720) within the RNB domain that moves away from the active site upon RNA binding (Supplementary information, Movie S4), as revealed earlier [10]. Therefore, Rrp44 undergoes an induced-fit enzymatic activation by its RNA substrates [24].…”

“…The sole RNase activity of the eukaryotic exosome comes from the tenth protein, npg www.cell-research.com | Cell Research Rrp44 (Dis3), which attaches to the bottom of the core complex. Rrp44 is a multi-domain protein with a processive hydrolytic exonuclease activity at its C-terminal region, which is homologous to bacterial RNase II, and a relatively weak endonuclease activity at its N-terminal region [10][11][12]. Previous structural studies have shown that Rrp44 specifically interacts with Rrp41, Rrp43 and Rrp45 of the core complex, aligning with the central channel's exit of the core complex [13][14][15].…”

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).…”

Validation of the orthogonal tilt reconstruction method with a biological test sample

“…We chose the exosome-a 398 kDa complex essential for RNA processing in yeast-as our test sample because (1) a Random Conical Tilt reconstruction of the sample, in negative stain, is already available ; (2) crystal structures of the core complex (lacking Rrp44) and the Rrp44 protein have been published (Liu et al, 2006;Lorentzen et al, 2008) and (3) by collecting data from the exact same grid used for the RCT reconstruction we could eliminate the effect of sample preparation as a variable in our results. The data presented here confirms the observations we originally made with synthetic data: initial models obtained with OTR are fully sampled in Fourier space (thus lacking artifacts) and can be directly used for refinement without further intervention by the user, allowing for the method to be automated.…”