The eukaryotic exosome is a ten subunit 3′ exoribonuclease complex responsible for many RNA processing and degradation reactions. How the exosome accomplishes this is unknown. We show that the PIN domain of Rrp44 is an endoribonuclease. The activity of the PIN domain prefers RNA with a 5′ phosphate, suggesting coordination of 5′ and 3′ processing. We also show that the endonuclease activity is important in vivo. Furthermore, the essential exosome subunit Csl4 does not contain any essential domains, but its zinc-ribbon domain is required for exosome-mediated mRNA decay. These results suggest that specific exosome domains contribute to specific functions, and that different RNAs interact with the exosome differently. The combination of an endoribonuclease and exoribonuclease activity appears to be a widespread feature of RNA degrading machines.

The exosome consists of a core of ten essential proteins that includes the ribonuclease Rrp44p and is present in both the cytoplasm and nucleus of eukaryotic cells. The cytoplasmic exosome has been extensively characterized in the budding yeast Saccharomyces cerevisiae and some characterization of its metazoan counterpart indicates that most functional aspects are conserved. These studies have implicated the cytoplasmic exosome in the turnover of normal cellular mRNAs, as well as several mRNA surveillance pathways. For this, the exosome needs a set of four proteins that do not partake in nuclear exosome functions. These cofactors presumably direct the exosome to specific cytoplasmic RNA substrates. Here, we review cofactors and functions of the cytoplasmic exosome and provide unanswered questions on the mechanisms of cytoplasmic exosome function.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

RNA degradation plays important roles for maintaining temporal control and fidelity of gene expression, as well as processing of transcripts. In Saccharomyces cerevisiae the RNA exosome is a major 3 ′ -to-5 ′ exoribonuclease and also has an endonuclease domain of unknown function. Here we report a physiological role for the exosome in response to a stimulus. We show that inactivating the exoribonuclease active site of Rrp44 up-regulates the iron uptake regulon. This up-regulation is caused by increased levels of reactive oxygen species (ROS) in the mutant. Elevated ROS also causes hypersensitivity to H 2 O 2 , which can be reduced by the addition of iron to H 2 O 2 stressed cells. Finally, we show that the previously characterized slow growth phenotype of rrp44-exo − is largely ameliorated during fermentative growth. While the molecular functions of Rrp44 and the RNA exosome have been extensively characterized, our studies characterize how this molecular function affects the physiology of the organism.

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.