Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex

Abstract

Trax-translin heteromers, also known as C3PO, have been proposed to activate the RNA-induced silencing complex (RISC) by facilitating endonucleolytic cleavage of the siRNA passenger strand. We report on the crystal structure of hexameric Drosophila C3PO formed by truncated translin and Trax, along with electron microscopic and mass spectrometric studies on octameric C3PO formed by full-length translin and Trax. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoRNase activity and interacts extensively with translin to form an octameric assembly. The catalytic pockets of Trax subunits are located within the interior chamber of the octameric scaffold. Truncated C3PO, like full-length C3PO, shows endoRNase activity that leaves 3'-hydroxyl-cleaved ends. We have measured the catalytic activity of C3PO and shown it to cleave almost stoichiometric amounts of substrate per second.

Figure 1

Overall structure of truncated hexameric C3PO, Trax-Translin interactions and an active center in Trax for endoribonuclease activity. (a) Lengths of wild-type Translin and Trax and truncated constructs that yielded crystals of C3PO. (b) Ribbon representation of truncated C3PO with two molecules of Trax colored in pink and four molecules of Translin colored in different shades of cyan. (c) Surface representation of truncated C3PO in the same view and color-coded as in (b). (d) Structural superposition of Trax (pink) and Translin (cyan) shown in ribbon representation. (e) Heterodimer of Trax and Translin as seen in the truncated C3PO structure (f) Homodimer of Translin positioned between the two heterodimers of Trax and Translin in the truncated C3PO structure. (g) Electrostatic surface and ribbon representation of Trax showing presence of three acidic residues (shown in box) along one face in the center of the Trax molecule. An enlarged view of this region is shown in a box. (h) Electrostatic surface representation of truncated C3PO showing location of active sites within the inner concave face (indicated by yellow arrows) for the endoribonuclease activity.

Figure 2

Mass spectra and simulated spectrum of the full-length C3PO. (a) Mass spectrum of the full-length C3PO at high collision energy (80 V). The full-length C3PO undergoes gas-phase dissociation such that free monomers of translin (blue hexagons) and Trax (yellow stars) are observed at low m/z (left inset) leaving 'stripped' complexes at high m/z (right inset). Two stripped complexes for the 6:2 (translin:Trax) complex are observed because either translin (pink squares) or Trax (beige squares) can dissociate. Similarly, for the 5:3 (translin:Trax) complex, stripped complexes without translin (light blue oval) or Trax (pink squares) are observed. (b) Mass spectrum showing the m/z region of a (6,600–9,200). Translin:Trax complexes with 6:2 (red squares) and 5:3 (blue ovals) stoichiometries are identified. A similar spectrum was obtained at a collision energy of 50 V. (c) Simulated mass spectrum of the 6:2 and 5:3 translin:Trax C3PO was generated automatically using an algorithm (F. Stengel, A.J. Baldwin, M.F. Bush, H. Lioe, N. Jaya et al., unpublished data) implemented in Python, which suggests the presence of 63% and 37% occurrence of these two species, respectively. The measured spectrum obtained at collision energy 50 V is overlaid on the observed spectrum.

Figure 3

Classification and averaging of negatively-stained particle images and fit of octameric C3PO into 3D EM map. (a) Montage of nine panels for comparison between reprojection of the reconstruction (image I) with the corresponding class average (image II) showing a representative particle in that class (image III). (b) Docking of the octameric full-length C3PO (Translin:Trax ratio of 6:2) model in the 3D EM map. Trax (pink) and Translin (cyan) molecules are shown in ribbon representation. Both 6:2 and 5:3 Translin:Trax models of C3PO could be fitted satisfactorily within the EM envelope. Top and side views related by a 90° rotation are shown in left and right panels, respectively.

Figure 4

Models of full-length octameric C3PO (6:2 and 5:3 Translin:Trax) based on the crystal structure of truncated hexameric C3PO (4:2 Translin:Trax). (a) Crystal structure of truncated hexameric C3PO (4:2 Translin:Trax) with Translin in cyan and Trax in pink. (b) Model of the full-length octameric C3PO (6:2 Translin:Trax) generated by addition of a homodimer of Translin (cyan) into the crystal structure of truncated C3PO. (c) Model of the full-length octameric C3PO (5:3 Translin:Trax) generated by addition of a heterodimer of Translin (cyan) and Trax (pink) into the crystal structure of truncated C3PO.

Figure 5

C3PO ribonuclease activity generates products with 2′ and 3′ hydroxyl termini and requires Glu123 and Glu126 residues in Trax. (a) Two 5′ ³²P-labelled 21-nt oligoribonucleotides were incubated with indicated recombinant C3PO complex and protein concentration: full-length (fl), truncated (t), and the catalytic mutant C3PO (m) in which two glutamic acid residues of Trax were converted to alanine (E123A, E126A). The reactions were stopped and reaction products were separated by 18% denaturing polyacrylamide gel electrophoresis. Alkaline hydrolysis treatment of RNA substrate generates products with 2′,3′-cyclic and 2′ and 3′ monophohosphate ends, which resolve into doublets towards the bottom of the gel. Doublet bands were bracketed, and the upper band represents fragments with 2′ or 3′ phosphate and the lower band the 2′,3′-cylic phosphate product; RNase T1 generated 3′ phosphate ends only. The mobility of C3PO-digested fragments was reduced compared to their 2′ or 3′ phosphorylated derivatives. (b) A chimeric DNA/RNA oligonucleotide: 5′ ³²pTATCG-AGGTGAACATCACGTACGCGGAAUACUUCGAAATGTCCGTTCGGT, containing 12 internal RNA residues (underlined), was incubated with indicated concentrations of C3PO. Half of the reaction solution was subjected to periodate oxidation and β-elimination reaction. For comparison, partial alkaline-hydrolysed RNA was first 3′-dephosphorylated using T4 polynucleotide kinase and then subjected to oxidation andβ-elimination. The identities of RNA and DNA residues are shown; italicised residues indicate that the 3′-end contains a phosphate due to β-elimination. Abbreviations: T1, partial RNase T1 digest; H, partial alkaline hydrolysis; In, input RNA, “…”, bands representing subsequent RNA bases towards the 3′ end of the oligonucleotide sequence.

Figure 6

C3PO endoribonucleolytic activity is length-dependent. (a), Recombinant C3PO complexes retain endoribonucleolytic cleavage of circularized RNA. Indicated concentrations of C3PO were incubated with a 25-nt linear or circular radiolabelled RNA containing a single guanosine residue. Circular RNA was validated by its reduced migration (arrows) upon linearising digestion by RNase T1, and its resistance to exonuclease T. (b), RNA length dependence of C3PO cleavage. Radiolabelled poly(GU) oligoribonucleotides ranging from 4 to 12 nt in length were subjected to full-length C3PO cleavage at indicated times. The identities of the shortest C3PO-digested fragments are shown. For assignment of cleavage products, the 12-nt substrate was subjected to partial alkaline hydrolysis or RNase T1 digestion, both followed by treatment with T4 PNK to remove 2′ and 3′ phosphate and 2′,3′ cyclic phosphate ends. Reactions were stopped and separated on an 18% denaturing polyacrylamide gel. Abbreviations: T1, partial RNase T1 digest; H, partial alkaline hydrolysis; E, partial Exonuclease T digest.