Dis3 is an evolutionarily conserved and essential enzyme [1
], most known for its association with the RNA surveillance complex, the exosome [3
]. Although Dis3 interacts with exosome proteins, the enzyme is functionally distinct from the complex. Dis3 is a homolog of the hydrolytic bacterial RNases, RNase II and RNase R [4
]. These enzymes share a conserved set of amino acid motifs that fold into a pocket housing the 3’ to 5’ exoribonuclease activity of the protein, the RNB domain [4
]. The budding yeast Saccharomyces cerevisiae
Dis3 (ScDis3) enzyme, from which all current biochemical knowledge of Dis3 has emerged, requires both monovalent and divalent ions for RNB-mediated catalysis [5
]. In vitro
, no other co-factors are necessary for activity [5
Unlike their prokaryotic counterparts, eukaryotic Dis3 family members contain an N-terminal extension that harbors an additional domain with endoribonuclease activity, the PIN domain [6
]. Requirements for PIN-mediated activity are not well established. Work related to the Dis3 PIN and RNB active sites has emerged from six studies of the yeast homolog [5
]. Recognition of two enzymatically active domains in Dis3 has made characterizing the ribonuclease activity of the full-length protein a challenge. For example, in vitro
, either the RNB alone or the PIN alone is sufficient for activity, and individual RNB and PIN mutations only abrogate their respective activities. This suggests a functional separation of active sites, where one activity does not necessarily rely on the other. However, point mutations in either the ScDis3 RNB or PIN domain result in rRNA processing defects in vivo
, suggesting that for some RNA metabolic events, the domains may work together. Presently, the interplay of these domains in the context of full-length Dis3 is poorly understood. In this regard, Dis3 could act on RNA substrates as an exoribonuclease, endoribonuclease, or both.
In addition to the PIN domain, the Dis3 N-terminus (~300 amino acids long) includes several domains that may contribute to the ribonuclease activities of the protein. All Dis3 homologs contain a C3 motif, or a putative iron-sulfur binding domain, and two OB folds, or oligonucleotide-binding folds [8
]. These domains have been shown to be important for cell growth in yeast [8
] as well as core exosome binding [9
], and nuclear targeting [12
]. A domain with homology to the mitotic cohesin STAG resides within the Drosophila
Dis3 N-terminus, but its function is unclear [12
]. A fundamental question is to determine how or whether these domains contribute to Dis3 ribonuclease activities.
Another challenge to understanding Dis3 ribonuclease activities is identification of their substrate specificities and reaction products. The full-length ScDis3 cleaves RNAs regardless of sequence or structure, indicating that the protein is a non-specific ribonuclease [3
]. The products of these reactions vary depending on substrate, but typically range in length from 2-5 nucleotides, resulting from either endo- or exoribonuclease activity. ScDis3 activity also releases 5’NMP products, a characteristic of exoribonuclease activity, and RNB point mutations eliminate this ability [5
Whether metazoan Dis3 homologs have similar activities, ionic requirements, substrate specificity, and reaction products has not been explored. To address this, we characterize Drosophila melanogaster Dis3 (dDis3). Specifically, we assess requirements for in vitro ribonuclease activity, the ability of the enzyme to degrade different RNA substrates, the contributions of distinct domains to activity, and the strength of the dDis3-core exosome interaction. As this study represents a first analysis of a metazoan Dis3, our findings help build a more complete picture of the general features of Dis3 ribonuclease activities.