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Structure of the Arabidopsis Thaliana DCL4 DUF283 Domain Reveals a Noncanonical Double-Stranded RNA-Binding Fold for Protein–Protein Interaction

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Structure of the Arabidopsis Thaliana DCL4 DUF283 Domain Reveals a Noncanonical Double-Stranded RNA-Binding Fold for Protein–Protein Interaction Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein–protein interaction HAINA QIN,1,5 FADING CHEN,1,2,5 XUELU HUAN,3,5 SATORU MACHIDA,1,2 JIANXING SONG,1,4 and Y. ADAM YUAN1,2 1Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore 2Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore 3NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456, Singapore 4Department of Biochemistry, National University of Singapore, Singapore 119260, Singapore ABSTRACT Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an a-b-b-b-a topology and resembles the structural similarity to the double- stranded RNA-binding domain. Notably, the N-terminal a helix of DUF283 runs cross over the C-terminal a helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing. Keywords: NMR structure; Dicer DUF283; miRNA processing; double-stranded RNA-binding fold; protein–protein interaction INTRODUCTION Argonaute are essential for the initiation and effect of RNA silencing, respectively (Voinnet 2005). RNA silencing is a small regulatory RNA-controlled and Dicers not only play the catalytic role to process revolutionary conserved gene regulation mechanism com- miRNAs/siRNAs, but also load these small regulatory RNAs prising a set of sequential core reactions (Zamore and into the RISC (Lee et al. 2004; Pham et al. 2004). In Haley 2005). First, Dicer-like enzyme processes primary Arabidopsis thaliana, there are four Dicer-like proteins, miRNA transcript (pri-miRNA) or long complementary namely DCL1–4. DCL1 plays the role for miRNA proces- double-stranded RNA into 21–24 base pairs (bp) small sing, whereas DCL2–4 proteins generate siRNAs with dis- regulatory RNA (Bernstein et al. 2001). Subsequently, the tinct sizes. DCL2 processes long dsRNAs into 22 bp, small RNA is loaded into RNA-induced silencing complex whereas DCL3 and DCL4 produce 24 bp siRNA and 21 bp (RISC) to pair with target mRNA for degradation or trans- siRNA, respectively (Qi et al. 2005). Therefore the struc- lation repression (Hammond et al. 2000; Nykanen et al. tural information of Dicer could provide the insightful in- 2001; Liu et al. 2004; Meister et al. 2004; Siomi and Siomi formation to understand the molecular mechanism of 2009). RNase III enzyme Dicer and RNase H enzyme Dicer initiated RNA silencing pathway. Dicer protein is a multiple-domain protein, which con- 5These authors contributed equally to this work. tains the helicase domain, DUF283 domain, PAZ domain, Reprint requests to: Jianxing Song, Department of Biological Sciences, two tandem RNase III domains, and two tandem dsRBD do- National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; e-mail: [email protected]; fax: (65)-67792486; or Y. Adam mains from the N-terminus to C-terminus (Fig. 1A). It was Yuan, Temasek Life Sciences Laboratory, National University of Singapore, proposed that the RNase III domain harbors the catalytic 1 Research Link, Singapore 117604, Singapore; e-mail: [email protected]; residues for small RNA processing (Zhang et al. 2004). The fax: (65)-68727007. Article published online ahead of print. Article and publication date recent crystal structure of human parasite Giardia Dicer are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1965310. provides the detailed molecular evidence to support the 474 RNA (2010), 16:474–481. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2010 RNA Society. Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Structure of the DCL4 DUF283 domain FIGURE 1. Overall structure of DCL4 DUF283. (A) Domain architecture of Arabidopsis thaliana DCL4. (B) Sequence alignment and secondary structure of Dicer DUF283. The aligned sequences (Swiss Protein ID) are in the order of DCL4_At, DCL1_At, DCL2_At, DCL3_At, and Dicer_Hs. The secondary structure diagram for DCL4_At is shown on top. The a-helices are colored in yellow, b-strands are colored in cyan. Conserved residues are shaded in cyan (80% similarity) and green (60% similarity), whereas essentially invariant residues are shaded in yellow. (C) Stereo view of the ensemble of eight lowest energy NMR structures of the DCL4 DUF283 domain (residues 651–752) and Ribbon representation of the DCL4 DUF283 domain. long standing hypothesis that Dicer harbors ‘‘one proces- In an effort to understand the functional role of the sing center’’ to cleave the bound pre-miRNA or long dsRNA DUF283 domain in Dicer-catalyzed RNA silencing path- into z19 bp small RNA duplex with 39-2-nucleotide (nt) way, we report here the first structure of the DUF283 overhangs (Macrae et al. 2006). Remarkably, the Giardia domain of the Arabidopsis Dicer-like 4 (DCL4) protein and Dicer structure adopts a hatchet-like architecture with the PAZ show that DUF283 adopts a double-stranded RNA-binding domain recognizing the 39-2-nt overhangs of bound dsRNAs, (dsRBD) fold for protein–protein interaction, which is in whereas the unique connecting a helix functions as a mo- contrast to bioinformatics prediction that DUF283 is lecular ruler to measure the distance from the dsRNA end a dsRBD fold for dsRNA binding (Dlakic´ 2006). We further (recognized by the PAZ domain) to the cleavage site (provided demonstrate that the DCL4 DUF283 domain selectively by the RNase III domains) (Macrae et al. 2006). Although binds to its designated partner, DRB4, whereas the DCL1 Giardia Dicer contains only the PAZ domain and two DUF283 domain selectively binds to its designated partner, RNase III domains, it displays robust dsRNA-processing HYL1, by in vitro pull-down assay, which suggest that activity (Macrae et al. 2006). By contrast, the removal of the Arabidopsis DUF283 domains probably play significant DUF283 domain, which is z100 amino acid positioning at roles for partner protein selection in small RNA processing. the N-terminus of the PAZ domain (Fig. 1A,B), from either human dicer or Drosophila DCR-1 abolished miRNA pro- RESULTS cessing activity (Lee et al. 2006; Ye et al. 2007). However, a slightly different DUF283 deletion construct in human The DUF283 domain resembles dsRNA-binding fold dicer shows little impact on pre-siRNA or pre-miRNA cleavage activity (Ma et al. 2008). Nevertheless, these data We have systematically screened z60 different Dicer DUF283 suggest that the DUF283 domain could be an essential constructs from multiple species with different fragment functional domain for Dicers from certain higher eukaryotes. lengths and tags to assess the expression levels and solubility www.rnajournal.org 475 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Qin et al. of the expressed proteins. The Arabidopsis DCL4 DUF283 aeolicus RNase III (PDBID:1RC7, Z score, 4.6, RMSD 3.7 construct (residues: 651–752) used in our experiments is A˚ ,62Ca) (Fig. 2A). The comparisons of these two proteins suitable for NMR determination because of its high ex- show structural similarity at their b-sheet and C-terminal pression level, solubility, and monomeric dispersion. a-helix regions. However, the N-terminal a-helix of DCL4-DUF283 has a highly dispersed HSQC spectrum DUF283 swings z30° back toward the C-terminal a helix, in solution characteristic of a well-folded protein. As such, hence the N- and C-termini of DUF283 are in close the NMR structure of DUF283 was successfully determined proximity, which is different from the bioinformatics by using NOE distance restraints derived from analyzing prediction (Dlakic´ 2006). 15N- and 13C-edited NOESY spectra; as well as dihedral angle restraints from TALOS prediction based on five The DUF283 domain is a noncanonical dsRBD chemical shifts. Figure 1C presents the superimposition of 10 selected DUF283 structures in solution with the lowest In addition to the different orientations of the N-terminal target functions. As detailed in Table 1, over the secondary a-helix between DUF283 and dsRBD (Fig. 2A), there are structure regions, the 10 structures are very similar, with significant structural deviations between them at the puta- RMS deviations of 1.50 A˚ for all atoms, 1.26 A˚ for heavy tive RNA-binding surfaces (Fig. 2B). All the canonical atoms and 0.57 A˚ for backbone atoms. Interestingly, it dsRBDs exampled by Xenopus laevis dsRBD2 have three appears that the C-terminal z18 residues (residues: 735– critical dsRNA-binding regions, referred to as regions 1, 2, 752) are relatively unstructured and poorly defined, having
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