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RRP6 from Trypanosoma brucei: crystal structure of the catalytic domain, association with EAP3 and activity towards structured and non-structured RNA substrates.

Barbosa RL, Legrand P, Wien F, Pineau B, Thompson A, Guimarães BG - PLoS ONE (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.

ABSTRACT
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.

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Purification of TbRRP6 constructions and complexes with TbEAP3.A) Schematic representation of the TbRRP6 constructions tested for recombinant expression. TbRRP6CAT includes the EXO and HRDC domains that constitute the catalytic core of the protein; TbRRP6ΔC, in addition to the EXO and HRDC, contains the N-terminal PMC2NT domain, which was shown to interact with Rrp47 in yeast [19]. TbRRP6Δsig comprises the entire protein except for a 19-residues N-terminal sequence identified as a signal peptide. B) SDS-PAGE analysis of the purified TbRRP6CAT (lane 1) and the co-purified complexes TbRRP6ΔC-EAP3, TbRRP6ΔC-EAP3ΔC1 and TbRRP6ΔC-EAP3ΔC2 (lanes 2, 3 and 4, respectively). The additional band observed during the purification of the complex TbRRP6ΔC-EAP3 (lane 2) corresponds to the degradation product of TbEAP3. The sizes of the molecular weight markers (M) are given in kDa. C) SEC-MALS analyses of TbRRP6CAT and the complex TbRRP6ΔC-EAP3ΔC1. The estimated molecular masses indicate that TbRRP6CAT is a monomer in solution while TbRRP6ΔC and TbEAP3ΔC1 form a heterodimer.
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pone-0089138-g002: Purification of TbRRP6 constructions and complexes with TbEAP3.A) Schematic representation of the TbRRP6 constructions tested for recombinant expression. TbRRP6CAT includes the EXO and HRDC domains that constitute the catalytic core of the protein; TbRRP6ΔC, in addition to the EXO and HRDC, contains the N-terminal PMC2NT domain, which was shown to interact with Rrp47 in yeast [19]. TbRRP6Δsig comprises the entire protein except for a 19-residues N-terminal sequence identified as a signal peptide. B) SDS-PAGE analysis of the purified TbRRP6CAT (lane 1) and the co-purified complexes TbRRP6ΔC-EAP3, TbRRP6ΔC-EAP3ΔC1 and TbRRP6ΔC-EAP3ΔC2 (lanes 2, 3 and 4, respectively). The additional band observed during the purification of the complex TbRRP6ΔC-EAP3 (lane 2) corresponds to the degradation product of TbEAP3. The sizes of the molecular weight markers (M) are given in kDa. C) SEC-MALS analyses of TbRRP6CAT and the complex TbRRP6ΔC-EAP3ΔC1. The estimated molecular masses indicate that TbRRP6CAT is a monomer in solution while TbRRP6ΔC and TbEAP3ΔC1 form a heterodimer.

Mentions: The genes encoding TbRRP6 (Tb927.4.1630) and TbEAP3 (Tb927.7.5460) were synthesized by GeneArt Gene Synthesis (Life Technologies). The corresponding TbRRP6 DNA sequences were subcloned into the pET28a vector (Novagen) to express TbRRP6Δsig (residues 20–736), TbRRP6ΔC (residues 20 to 540) and TbRRP6CAT (residues 176 to 540) constructs in fusion with a C-terminal His-tag. TbEAP3 coding sequence was subcloned into the pET21a vector (Novagen) to express the full-length TbEAP3 protein and the truncated forms TbEAP3ΔC1 (residues 1 to 183) and TbEAP3ΔC2 (residues 1 to 144) (Figures 1A and 2A).


RRP6 from Trypanosoma brucei: crystal structure of the catalytic domain, association with EAP3 and activity towards structured and non-structured RNA substrates.

Barbosa RL, Legrand P, Wien F, Pineau B, Thompson A, Guimarães BG - PLoS ONE (2014)

Purification of TbRRP6 constructions and complexes with TbEAP3.A) Schematic representation of the TbRRP6 constructions tested for recombinant expression. TbRRP6CAT includes the EXO and HRDC domains that constitute the catalytic core of the protein; TbRRP6ΔC, in addition to the EXO and HRDC, contains the N-terminal PMC2NT domain, which was shown to interact with Rrp47 in yeast [19]. TbRRP6Δsig comprises the entire protein except for a 19-residues N-terminal sequence identified as a signal peptide. B) SDS-PAGE analysis of the purified TbRRP6CAT (lane 1) and the co-purified complexes TbRRP6ΔC-EAP3, TbRRP6ΔC-EAP3ΔC1 and TbRRP6ΔC-EAP3ΔC2 (lanes 2, 3 and 4, respectively). The additional band observed during the purification of the complex TbRRP6ΔC-EAP3 (lane 2) corresponds to the degradation product of TbEAP3. The sizes of the molecular weight markers (M) are given in kDa. C) SEC-MALS analyses of TbRRP6CAT and the complex TbRRP6ΔC-EAP3ΔC1. The estimated molecular masses indicate that TbRRP6CAT is a monomer in solution while TbRRP6ΔC and TbEAP3ΔC1 form a heterodimer.
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Related In: Results  -  Collection

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pone-0089138-g002: Purification of TbRRP6 constructions and complexes with TbEAP3.A) Schematic representation of the TbRRP6 constructions tested for recombinant expression. TbRRP6CAT includes the EXO and HRDC domains that constitute the catalytic core of the protein; TbRRP6ΔC, in addition to the EXO and HRDC, contains the N-terminal PMC2NT domain, which was shown to interact with Rrp47 in yeast [19]. TbRRP6Δsig comprises the entire protein except for a 19-residues N-terminal sequence identified as a signal peptide. B) SDS-PAGE analysis of the purified TbRRP6CAT (lane 1) and the co-purified complexes TbRRP6ΔC-EAP3, TbRRP6ΔC-EAP3ΔC1 and TbRRP6ΔC-EAP3ΔC2 (lanes 2, 3 and 4, respectively). The additional band observed during the purification of the complex TbRRP6ΔC-EAP3 (lane 2) corresponds to the degradation product of TbEAP3. The sizes of the molecular weight markers (M) are given in kDa. C) SEC-MALS analyses of TbRRP6CAT and the complex TbRRP6ΔC-EAP3ΔC1. The estimated molecular masses indicate that TbRRP6CAT is a monomer in solution while TbRRP6ΔC and TbEAP3ΔC1 form a heterodimer.
Mentions: The genes encoding TbRRP6 (Tb927.4.1630) and TbEAP3 (Tb927.7.5460) were synthesized by GeneArt Gene Synthesis (Life Technologies). The corresponding TbRRP6 DNA sequences were subcloned into the pET28a vector (Novagen) to express TbRRP6Δsig (residues 20–736), TbRRP6ΔC (residues 20 to 540) and TbRRP6CAT (residues 176 to 540) constructs in fusion with a C-terminal His-tag. TbEAP3 coding sequence was subcloned into the pET21a vector (Novagen) to express the full-length TbEAP3 protein and the truncated forms TbEAP3ΔC1 (residues 1 to 183) and TbEAP3ΔC2 (residues 1 to 144) (Figures 1A and 2A).

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.

View Article: PubMed Central - PubMed

Affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.

ABSTRACT
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.

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