Ribonucleases (RNases) degrade or process RNA by endonucleolytic or exonucleolytic mechanisms. These activities are vital to cellular function, as reduction of RNase activities can severely impact cell growth, division and viability. Moreover, RNases are evolutionarily conserved in archaea, eubacteria and eukaryotes and hence represent a crucial and fundamental feature of all living cells.
Dis3 is an essential, conserved RNase that possesses 3′ → 5′ exo- and endoribonuclease activites (1
). A homolog of the eubacterial RNase II/R, Dis3 has been implicated in the processing and degradation of ribosomal RNAs [rRNA (3–8
)], messenger RNAs [mRNA (5
)] and transfer RNAs [tRNA (10
)]. The major structural difference between RNase II/R and Dis3 is an ~300 amino acid N-terminal extension that contains multiple bioinformatically identified domains [(2
), A]. These include a conserved set of three cysteine residues that resemble an iron–sulfur cluster motif [referred to here as C3 (7
)], a PIN endoribonuclease domain (6–8
), and, in the Drosophila melanogaster
protein, a motif with homolgy to the cohesin protein STAG (12
). Both RNase II/R and Dis3 harbor two N-terminal oligonucleotide-binding (OB) fold domains, an RNB exoribonuclease domain and a C-terminal S1 RNA-binding domain (2
). Most Dis3 homologs have a C-terminal extension that contains additional, uncharacterized variant sequences and, in the Drosophila
homolog, a nuclear localization sequence [NLS, (12
)]. Few of these domains have been unequivocally demonstrated to have functional relevance to Dis3 activity, localization or interactions.
Figure 1. N-terminal dDis3 domains are necessary and sufficient for in vitro ribonuclease activity. (A) Schematic of full-length D. melanogaster Dis3. (B) Recombinant MBP-Dis3 proteins used in in vitro ribonuclease assays. Dis3 proteins were purified and quantified (more ...)
Analyses regarding Dis3 structure and function have been primarily done in yeast cells and/or using recombinant yeast polypeptides. Thus, it is not known if the majority of observed domain functions are conserved in multicellular eukaryotes. Studies of Saccharomyces cerevisiae
Dis3 have revealed that mutations in the N-terminal C3 domain impede cell growth, but for unknown reasons (7
). Mutations to conserved residues in the PIN or RNB domains result in loss of endoribonuclease or exoribonuclease activities, respectively (5–8
). These analyses indicate the PIN and RNB domains of yeast Dis3 contain ribonuclease active sites, although it is not known how other domains in the protein contribute to these activities. Consistent with these observations, the RNB domains of Drosophila
Dis3 and bacterial homologs RNase II/R alone contain exoribonuclease activity, thus Dis3 exoribonuclease activity is conserved (13–15
). However, it is not known if PIN endoribonuclease activity is conserved in Dis3 homologs. Finally, N-terminal domains appear to be important for localization of Drosophila
), but the relationship of the Dis3 N-terminus to its subcellular distribution is largely unknown.
Although Dis3 has been shown to function independently in vitro
, it was initially co-purified in a multiprotein complex (where it was termed Rrp44) of exoribonucleases called the exosome (3
). The original exosome core, thought to assemble and function in the nucleus and cytoplasm, contains RNase PH subunits Rrp41/Ski6, Rrp42, Rrp43, Rrp45, Rrp46 and Mtr3, and S1 domain subunits Rrp4, Rrp40 and Csl4 (16
). In S. cerevisiae
, the N-terminal PIN domain of Dis3 is responsible for interactions with these proteins (8
). N-terminal domains also appear to be important for Drosophila
Dis3 interactions with the exosome (12
), although the exact interacting domain has not been identified.
The exosome core associates with additional proteins, including the RNase D homolog Rrp6, and the nuclear exosome cofactor Rrp47/Lrp1. However, Rrp6 and Rrp47 function independently of the exosome core as well (18–20
). In this regard, there is significant biochemical evidence that exosome polypeptides assemble into multiple distinct complexes, several of which lack many subunits, including Dis3 (21
). Dis3 itself is found in complexes independent of the exosome (23–26
). The exact number of Dis3 complexes, the site(s) of their assembly and disassembly, and the domains of Dis3 that mediate specific protein–protein interactions remain largely unknown.
In this work, we focus on the contributions of D. melanogaster Dis3 N-terminal domains to functions in vitro and in vivo. First, we examine if the N-terminal endoribonuclease activity reported for S. cerevisiae Dis3 is conserved in the Drosophila Dis3 enzyme. We also use truncation and point mutants to explore the contributions of the dDis3 N-terminus to its subcellular distribution, and interactions with core exosome subunits, dRrp6 and dImportin-α3. Our study reveals novel features of the dDis3 N-terminus that are functionally relevant and hence of general and broad importance to exosome-mediated RNA metabolic pathways and mechanisms.