Bioinformatics Advance Access originally published online on September 5, 2006
Bioinformatics 2006 22(22):2711-2714; doi:10.1093/bioinformatics/btl468
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DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold
Department of Microbiology, Montana State University Bozeman, MT 59717, USA
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ABSTRACT |
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 TOP ABSTRACT 1 INTRODUCTION2 METHODS3 RESULTS AND DISCUSSIONREFERENCES |
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Two RNases, Dicer and Argonaute, are at the heart of the RNA interference (RNAi) molecular machinery responsible for gene silencing. Both RNases contain multiple domains, most of which have been characterized or have functions that can be predicted based on sequence comparisons. However, Dicers of higher eukaryotes contain the domain known as DUF283 which at present has no assigned role. Using sensitive profile–profile comparisons, we detected a divergent double-stranded RNA-binding domain coinciding with the DUF283 of Dicer. This finding has potential implications regarding the mechanistic role of Dicer in RNAi.
Contact: mdlakic{at}montana.edu
Supplementary information: Supplementary images are available at Bioinformatics online.
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1 INTRODUCTION |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS AND DISCUSSIONREFERENCES |
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RNA interference (RNAi) is an evolutionarily conserved mechanism for gene silencing using double-stranded RNA (dsRNA) molecules known as small interfering RNAs (siRNAs). Two nucleases, an RNase III enzyme Dicer and RNase H enzyme Argonaute, are essential for the initiation and effector phases of RNAi, respectively (Bernstein et al., 2001; Hammond et al., 2001). Recent structural and biochemical studies provided insights into functions of both enzymes (reviewed in Collins and Cheng, 2005; Hammond, 2005), yet many questions remain regarding the mechanistic aspects of RNAi (Tomari and Zamore, 2005). For example, Dicers of higher eukaryotes contain multiple protein domains for which the general function is known or can be predicted with confidence (Bernstein et al., 2001), yet the function is unknown for the so-called Domain of Unknown Function 283 (DUF283). DUF283 is classified in PFAM (Bateman et al., 2004) as a distinct domain based on its sequence conservation among Dicer-like proteins. However, even the sensitive sequence profile tools, such as HMMer and PSI-BLAST, failed to assign any function to this domain as it lacks clear similarity to characterized proteins. We used recently developed methods for profile–profile comparisons to show that DUF283 has a fold similar to the double-stranded RNA-binding domain (dsRBD). This finding is discussed in the light of currently unanswered questions about the mechanistic role of Dicer proteins.
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2 METHODS |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS AND DISCUSSIONREFERENCES |
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The consensus sequences of 8183 Pfam 19.0 families were extracted from their profile hidden Markov models (HMMs; Bateman et al., 2004). The PDB-derived collection (as of March, 2006) was clustered at 95% identity, and both groups of sequences were used as queries for the Target2K procedure (Karplus et al., 2005), which automatically searches the protein database and builds an alignment of identified homologs. Predicted secondary structure (Jones, 1999) was added to the alignments to generate profile HMMs (Soding, 2005). The principal difference between these databases and the ones distributed on the HHpred server (Soding et al., 2005) is that the alignments are, on average, more accurate yet include fewer sequences. Consensus sequence for DUF283 was also submitted to the 3D-Jury meta server (Ginalski et al., 2003).
Model building was done using MODELLER 8v2 (Eswar et al., 2003) and the side-chains were rebuilt using Dunbrack's backbone-dependent rotamer library (Canutescu et al., 2003). All models were evaluated by Prosa (Sippl, 1993), Verify3D (Luthy et al., 1992) and ProQ/MaxSub (Wallner and Elofsson, 2003).
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3 RESULTS AND DISCUSSION |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS AND DISCUSSIONREFERENCES |
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The development of novel computational methods in recent years has stretched the limits of identifying meaningful biological similarities from remote protein homology (Petrey and Honig, 2005). To search for distant relatives of DUF283, we used one such method (Soding, 2005) that employs pairwise alignments of HMMs. At present, this approach offers arguably the best combination of sensitivity needed to detect subtle similarities and the speed required to scan large databases of protein families (Soding, 2005). The consensus sequence of DUF283 (Pfam code PF03368) was extracted from the HMM profile deposited in Pfam (Bateman et al., 2004) and was used to generate the profile HMM of DUF283 as described above. We compared this HMM to protein families in Pfam and the search yielded significant similarity to the dsRNA binding motif (PF00035; E = 4.9 x 10–7). This finding was confirmed when comparing DUF283 profile HMM to known protein structures in PDB, as >10 significant matches to dsRNA binding domains (dsRBDs) were detected with E-values between 9 x 10–7 and 1 x 10–3. The credibility of this prediction is strengthened by an excellent match between predicted secondary structure of DUF283 (Jones, 1999) and the experimentally determined secondary structure of dsRNA binding proteins (ss_pred and ss_dssp lines in Fig. 1, respectively). Finally, the consensus sequence of DUF283 was submitted to the 3D-Jury server (http://bioinfo.pl/Meta/), which returned significant matches (scores >50) to three dsRBD-containing proteins (Ginalski et al., 2003).
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Due to low target-template sequence identity (<20%), we had to use a trial-and-error strategy for building 3D models of human and Drosophila's DUF283. After each cycle of model building and evaluation, the alignments were changed manually in regions that had unfavorable energy as judged by Prosa scores. The final model of Drosophila's DUF283 (Fig. 1) had the evaluation scores that are consistent with good to very good models: –4.57 for Prosa (Sippl, 1993); 19.49 for Verify3D (Luthy et al., 1992); 6.85 for ProQ and 0.72 for MaxSub (Wallner and Elofsson, 2003). In addition, the overall fold of the model is classified as correct based on the pG score of 0.91 (Sanchez and Sali, 1998).
dsRBD proteins adopt an 
-ß-ß-ß- topology that preferentially binds dsRNA over ssRNA or dsDNA, although without much sequence specificity (Ryter and Schultz, 1998). dsRBD is commonly found in many proteins involved in RNA processing (Doyle and Jantsch, 2002), including all three classes of RNase III enzymes (Hammond, 2005). Bacterial RNase III enzymes (Class I) contain one dsRBD and one RNase III domain and they form highly symmetric homodimers (Zhang et al., 2004; Gan et al., 2006). Dicers of higher eukaryotes, on the other hand, contain two joined RNase III domains that form an intramolecular dimer (Zhang et al., 2004), and have only one canonical dsRBD (Class III). Given the diversity of dsRNAs in eukaryotic cells, it is not clear how a single dsRBD could provide Dicers with distinct specificity for genuine siRNAs. Part of this functionality may be supplied by the PAZ domain (Lingel et al., 2004; Ma et al., 2004), or in trans by Dicer's binding partners that contain dsRBDs (Tabara et al., 2002). Our finding provides an additional avenue for future experiments aimed at better understanding of Dicer's binding specificity.
In a recent article, MacRae et al. (2006) presented the structure of Dicer from Giardia intestinalis. This seminal work provided the platform for understanding dsRNA processing that takes place during the initiation phase of RNAi. MacRae et al. (2006) suggested, based on ‘low but consistent sequence similarity’ between the N-terminal platform domain (NTPD) of Giardia Dicer and DUF283 (shown in their Supplementary Figure 4), that these two domains have similar structures. However, we find no proof for this sequence similarity in terms of statistical significance. For example, profile HMM of DUF283 does not match any part of Giardia Dicer even when the E-value is raised up to 100. In retrospect, it appears that the homology inferred by MacRae et al. was derived, at least in part, from the global similarity between these two domains: the NTPD of Giardia Dicer has a ß-ß-ß-ß--fold, while DUF283 is predicted to adopt the canonical -ß-ß-ß--fold found in all dsRBDs. Despite similar topologies, however, these two domains are structurally unrelated since their secondary structure elements have different spatial arrangements.
What could be the function of this divergent dsRBD coinciding with DUF283? In addition to their most common role in dsRNA binding, some dsRBDs have additional functions such as dimerization (Hitti et al., 2004) and nuclear localization (Eckmann et al., 2001). To gain insight into potential function of DUF283, we compared surface conservation of DUF283 and dsRBD proteins. The seed alignment of dsRBD from Pfam (Bateman et al., 2004) was used as an input for the ConSurf server (Landau et al., 2005). Residue conservation from this analysis was converted to colors and mapped onto the dsRBD structure from 1UIL. As expected, the RNA-binding face of dsRBD proteins contains the majority of highly and moderately conserved residues (colored in red and green, respectively, in Supplementary Figure 1A). Next we merged DUF283 and dsRBD alignments (Edgar and Sjolander, 2004), reasoning that this combined alignment would have a different pattern of sequence conservation if DUF283 homologs were unrelated to dsRBD proteins. The combined alignment was mapped onto the 3D model of DUF283 (Supplementary Figure 1B) and showed very similar pattern of surface conservation to dsRBD alone. This analysis provides a strong argument that the surface of DUF283 is similar to the majority of dsRBD proteins and could bind RNA. There are, however, several conserved residues in DUF283, e.g. the nearly invariant cysteines at positions 833 and 897 in Drosophila's Dicer, which have no equivalents in dsRBD proteins. These cysteines are mentioned as potential zinc ligands in Pfam annotation of DUF283, yet in our 3D model they are too far apart to be involved in metal coordination or disulphide bond formation (shown as sticks in Supplementary Figure 1B). These residues could be brought within interacting distance only if the packing of -helices in DUF283 is significantly different from our model.
Dicers not only generate siRNAs, but also accompany them into the RNA-induced silencing complex (RISC) (Lee et al., 2004; Pham et al., 2004). It is thought that the polarity of Dicer processing may influence the subsequent strand selection, as only one of the two strands of the siRNA will be used by the RISC (reviewed in Sontheimer, 2005). Therefore, any asymmetric interaction(s) between Dicer and siRNAs could establish the order and directionality of processing steps in RISC. In particular, this may determine from which end the dsRNA is unwound, leading to the incorporation of the ‘guide’ strand into RISC and the disposal of the ‘passenger’ strand (reviewed in Sontheimer, 2005; Tomari and Zamore, 2005). It has been shown that the relative thermal stability of base pairing at each end of the siRNA duplex provides this asymmetric signal, or at least part of it (Khvorova et al., 2003; Schwarz et al., 2003). It is not clear, however, whether Dicer recognizes the asymmetry of its substrate alone or in combination with other dsRNA-binding proteins (reviewed in Tomari and Zamore, 2005). In a scenario where Dicer does not need a protein partner for siRNA processing and subsequent strand selection, subtle differences in double-stranded quality of siRNA ends could be recognized by DUF283 and dsRBD; this hypothesis can be tested by exchanging the positions of the two domains. If, however, Dicer needs a dsRNA-binding partner for this task, DUF283 could instead serve as a dimerization domain. It has been documented that Dicers of Drosophila and human form heterodimers with TRBP/Loquacious, both of which contain three dsRBDs and are needed for proper microRNA (miRNA) maturation and subsequent assembly into RISC (Chendrimada et al., 2005; Forstemann et al., 2005; Haase et al., 2005). In addition, Drosophila's Dicer-2 forms a heterodimer with dsRBD protein R2D2, which in turn orients the heterodimers on the siRNA duplex (Tomari et al., 2004).
RNase III domains of Dicer form an intramolecular dimer and likely cleave RNA similarly to intermolecular dimers of bacterial RNase III (Zhang et al., 2004). However, fine details of these two ribonucleoprotein complexes must be different because Dicers have no dsRBD at the C-terminus of the first RNase III domain. Instead, DUF283 is positioned at the N-terminus of the first RNase III domain, creating an arrangement that additionally contributes to the asymmetry of the resulting protein–RNA complex. We therefore propose that DUF283 is involved in siRNA/miRNA strand selection, be it by recognizing the asymmetry of RNA duplexes directly or by recruiting another dsRBD protein. It will be interesting to examine how the recognition of siRNA/miRNA duplexes is transmitted to the adjacent helicase domain to aid correct strand selection for the RISC assembly.
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Acknowledgments |
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This work was supported in part by NIH Grant P20 RR16455-06 from the INBRE-BRIN Program of the National Center for Research Resources. Funding to pay the Open Access publication charges for this article was provided by NIH Grant P20 RR16455-06.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Golan Yona
Received on June 14, 2006; revised on August 7, 2006; accepted on August 29, 2006
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