Poly(A) Tail-dependent Exonuclease AtRrp41p fromArabidopsis thaliana Rescues 5.8 S rRNA Processing and mRNA Decay Defects of the Yeast ski6 Mutant and Is Found in an Exosome-sized Complex in Plant and Yeast Cells

Abstract

Eukaryotic 335 exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 335 exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 335 mRNA degradation defects of the yeast ski6 -100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6 -100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.A large number of eukaryotic RNA species require 3Ј35Ј exonucleolytic activities either for processing from their respective precursor forms or for their turnover. In yeast, a large, ϳ300 -400 kDa complex, the exosome, mediates many of these reactions (2, 21; for review, see Ref. 26). Two major forms of the exosome, nuclear and cytoplasmic, have been identified, each containing at least 10 common "core" components, Rrp4p, Rrp40p-Rrp46p, Mtr3p, and Csl4p proteins. In addition, Rrp6p appears to be associated only with the nuclear form of the exosome. Furthermore, RNA helicases Dob1p and Ski2p are required for at least some activities of the nuclear and cytoplasmic forms of the exosome, respectively, although their physical associations with it have not been documented.Interestingly, all but one of the core exosome subunits either have been shown to be 3Ј35Ј exonucleases in vitro or were predicted to have such activity, based on sequence similarity to known exonucleases. Six of the exosome components (Rrp41p, Rrp42p, Rrp43p, Rrp45p, Rrp46p, and Mtr3p) are homologous to the Escherichia coli RNase PH, and the recombinant yeast Rrp41p has a phosphorolytic, processive 3Ј35Ј exonuclease activity (21). Rrp4p and Rrp40p are homologous to each other and, along with Csl4p, contain a predicted S1 RNA binding motif. Recombinant Rrp4p is a hydrolytic, distributive 3Ј35Ј exonuclease. Rrp44p is homologous to the E. coli RNase R, of the RNase II family, and exhibits a hydrolytic, processive mode of RNA degradation. Finally, Rrp6p is a member of the RNase D family, and the recombinant Rrp6p has a...

The Pyrococcus Exosome Complex

“…These data combined with the results presented here suggest that the RNases PH ring of the exosome would be active only when all of its components are assembled together. In the case of eukaryotic Rrp41p, however, the isolated protein showed RNase activity (41,42). Further biochemical characterization of the eukaryotic exosome is necessary to establish the relationship with its archaeal counterpart.…”

The exozyme model: A continuum of functionally distinct complexes

“…A complete expanded function table and citation index is included in Supplemental Tables S1 and S2. Burkard and Butler 2000;Chekanova et al 2000Chekanova et al , 2002Estevez et al 2001). The most striking difference occurs in rrp6D yeast strains, which accumulate a specific 5.8S + 27 precursor (Allmang et al 1999a,b;Burkard and Butler 2000).…”

Finding the Exosome

“…Archeae and plants exhibit exonuclease activity mediated by RNase PH related subunits [14][15][16] , but this does not appear to be the case for other eukaryotic systems tested.…”