RRP6 from Trypanosoma brucei: crystal structure of the catalytic domain, association with EAP3 and activity towards structured and non-structured RNA substrates.
Bottom Line: RRP6 is a 3'-5' exoribonuclease associated to the eukaryotic exosome, a multiprotein complex essential for various RNA processing and degradation pathways.TbRRP6 was able to degrade single and double-stranded RNAs and also RNA substrates containing stem-loops including those with 3' stem-loop lacking single-stranded extensions.Finally, association with TbEAP3 did not significantly interfere with the TbRRP6 catalytic activity in vitro.
Affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
RRP6 is a 3'-5' exoribonuclease associated to the eukaryotic exosome, a multiprotein complex essential for various RNA processing and degradation pathways. In Trypanosoma brucei, RRP6 associates with the exosome in stoichiometric amounts and was localized in both cytoplasm and nucleus, in contrast to yeast Rrp6 which is exclusively nuclear. Here we report the biochemical and structural characterization of T. brucei RRP6 (TbRRP6) and its interaction with the so-called T. brucei Exosome Associated Protein 3 (TbEAP3), a potential orthologue of the yeast Rrp6 interacting protein, Rrp47. Recombinant TbEAP3 is a thermo stable homodimer in solution, however it forms a heterodimeric complex with TbRRP6 with 1∶1 stoichiometry. The crystallographic structure of the TbRRP6 catalytic core exposes for the first time the native catalytic site of this RNase and also reveals a disulfide bond linking two helices of the HRDC domain. RNA degradation assays show the distributive exoribonuclease activity of TbRRP6 and novel findings regarding the structural range of its RNA substrates. TbRRP6 was able to degrade single and double-stranded RNAs and also RNA substrates containing stem-loops including those with 3' stem-loop lacking single-stranded extensions. Finally, association with TbEAP3 did not significantly interfere with the TbRRP6 catalytic activity in vitro.
Mentions: Despite extensive trials, we were not able to crystallize any of the TbEAP3 variants or the complexes TbRRP6ΔC-EAP3ΔC1 and TbRRP6ΔC-EAP3ΔC2. The TbRRP6CAT construct crystallized in a sea urchin-like form composed of very thin needles. The crystals showed to be very hard to reproduce and to optimize and a single data set could be collected from an eventual protuberant needle. TbRRP6CAT crystal structure was refined at 2.4 Å resolution to final Rfactor/Rfree of 16%/22%, respectively (Table 1). The model covers residues 176 to 541 and includes 185 solvent molecules. The polypeptide chain was clearly defined by the electron density, except the residues 416 to 423 that could not be modeled. The 3D structure of the RRP6 catalytic domain was previously described for the yeast and human counterparts , . TbRRP6CAT shares 41% and 40% of sequence identity with the corresponding catalytic core of the yeast and human proteins respectively and, as expected, conserves their overall architecture. The EXO domain consists of a classical α/β fold composed by a six-stranded β-sheet flanked by α-helices and the HRDC domain is constituted of six α-helices (Figure 3A). Superposition of the T. brucei RRP6 structure with the human and yeast orthologues results in RMSD of 1.38 Å for 343 C-alpha atoms aligned and 1.42 Å for 333 C-alpha aligned, respectively. As previously described ,  the EXO and HRDC domains are connected by a linker. Comparison of the structures of the yeast and human orthologues showed that the longer linker of yeast Rrp6 narrows the active site entrance which was proposed to affect the ability of the yeast enzyme to degrade structured RNA substrates . The T. brucei RRP6 structure shows a closer match to the human RRP6, both proteins presenting a shorter linker and a more accessible active site (Figure 3B).