Contribution of domain structure to the RNA 3′ end processing and degradation functions of the nuclear exosome subunit Rrp6p

Abstract

The 3-5 riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3 ends of 5.8S rRNA, sno-and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3 ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.

Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6

“…Rrp6 contains three domains required for nuclear localization, exoribonuclease activity, and RNA substrate specificity (43). Although a number of mRNAs are deregulated in the absence of Rrp6 (Dataset S2) it seems that protein-coding transcripts are recognized by the protein as substrates only in mutants that fail to export them from the nucleus (44).…”

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

“…5 Structurefunction studies of Rrp6 proteins with point mutations in the exonuclease domain confirmed the two-metal ion mechanism and suggested that, like the exonuclease domains of DNA polymerases, Rrp6 utilizes a phenylalanine to stabilize the hydroxyl anion intermediate activated for phosphodiester bond cleavage. 6,7 Unlike Dis3/Rrp44, whose activity is attenuated by interaction with Exo9, Rrp6 retains its characteristic properties in the Exo11 complex. 8 Rrp6 contains two HRDC (Helicase RNaseD Cterminal) domains, only one of which was predicated by sequence homology.…”

Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants

“…The D238A single amino acid substitution disrupts the coordination of metal ions in the active site whereas the Y361A mutation prevents the activation of the hydrolytic water and thereby presumably abolishes exonucleolytic activity of the enzyme, while the D457A mutation in the conserved helicase and RNase D Cterminal (HRDC) domain alters protein interdomain contacts, possibly affecting coordination of the RNA substrate (Phillips and Butler 2003;Midtgaard et al 2006). In addition, deletion of the first 128 amino acids of Rrp6p has been shown to disrupt its interaction with Rrp47p (Stead et al 2007).…”