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Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

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Model of NcsA activity and sampylation (panel A) as well as its association with protein partners (panel B).Panel A, NcsA is proposed to catalyze the formation of 2-thiouridine (s2U) at the wobble uridine position of tRNAs specific for lysine (tRNALysUUU), glutamate (tRNAGluUUC), and glutamine (tRNAGlnUUG) via an adenylated tRNA intermediate using thiocarboxylated SAMP2 as a source of activated sulfur. The E1-like UbaA adenylates the C-terminal α-carboxylate group of SAMP2. This modification readies the Ub-fold SAMP2 for either thiocarboxylation via an enzyme (cysteine desulfurase or rhodanese) catalyzed persulfide sulfur or protein modification via formation of a UbaA-SAMP2 thioester intermediate. Polymeric chains of SAMP2 are formed on an NcsA lysine residue via isopeptide linkages that are cleaved by HvJAMM1 protease. Whether additional factors are needed to provide specificity to the sampylation system is unclear, as E2 and E3 homologs are not predicted based on genome sequence. Panel B, NcsA is found isopeptide linked to SAMP2 and non-covalently associated with various proteins, as noted by dotted red lines. NcsA partners include the E1-like UbaA and Ub-fold SAMP2 of the tRNA thiolation and sampylation pathways. NcsA is also found associated with EF-1α that binds aminoacylated-tRNAs and mediates translation elongation, PAN-A/1 (an AAA+ATPase associated with energy-dependent proteolysis by proteasomes (20S core particles or CPs) and protein remodeling, and the β-CASP ribonuclease homolog of the aCPSF1 family thought to cleave mRNA and/or tRNA. RNAP, RNA polymerase.
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pone-0099104-g008: Model of NcsA activity and sampylation (panel A) as well as its association with protein partners (panel B).Panel A, NcsA is proposed to catalyze the formation of 2-thiouridine (s2U) at the wobble uridine position of tRNAs specific for lysine (tRNALysUUU), glutamate (tRNAGluUUC), and glutamine (tRNAGlnUUG) via an adenylated tRNA intermediate using thiocarboxylated SAMP2 as a source of activated sulfur. The E1-like UbaA adenylates the C-terminal α-carboxylate group of SAMP2. This modification readies the Ub-fold SAMP2 for either thiocarboxylation via an enzyme (cysteine desulfurase or rhodanese) catalyzed persulfide sulfur or protein modification via formation of a UbaA-SAMP2 thioester intermediate. Polymeric chains of SAMP2 are formed on an NcsA lysine residue via isopeptide linkages that are cleaved by HvJAMM1 protease. Whether additional factors are needed to provide specificity to the sampylation system is unclear, as E2 and E3 homologs are not predicted based on genome sequence. Panel B, NcsA is found isopeptide linked to SAMP2 and non-covalently associated with various proteins, as noted by dotted red lines. NcsA partners include the E1-like UbaA and Ub-fold SAMP2 of the tRNA thiolation and sampylation pathways. NcsA is also found associated with EF-1α that binds aminoacylated-tRNAs and mediates translation elongation, PAN-A/1 (an AAA+ATPase associated with energy-dependent proteolysis by proteasomes (20S core particles or CPs) and protein remodeling, and the β-CASP ribonuclease homolog of the aCPSF1 family thought to cleave mRNA and/or tRNA. RNAP, RNA polymerase.

Mentions: Based on this study, we propose a model in which NcsA is covalently modified at lysine residue(s) by isopeptide linked chains of the ubiquitin-fold SAMP2, through a mechanism that requires the ubiquitin-activating E1 enzyme homolog UbaA (Figure 8A). While polymeric chains of SAMP2/3 were previously detected in the Hfx. volcanii proteome by MS/MS [13], [25], it was unclear whether or not these chains were anchored to protein targets. The prokaryotic ubiquitin-like protein (Pup) of mycobacteria is not known to form polymeric chains [32]. Thus, NcsA is the first example of a target protein that is isopeptide-linked to polymeric chains of an ubiquitin-like protein modifier in prokaryotic cells. While the lysine residue (Lys204) of NscA that was found modified by poly-SAMP2 is not highly conserved among archaea and appears restricted to species of Haloferax and Natrialba, Lys204 is predicted to be in a unstructured region of NcsA (highlighted in Figure 1) that has undergone large amino acid insertions in some species (e.g., Haloarcula vallismortis GI:490650014). Interestingly, the tRNA thiolase of T. thermophilus, TtuA, was recently demonstrated to be isopeptide linked at multiple lysine residues (including Lys137, 226 and 229; highlighted in Figure 1) to the ubiquitin-like protein modifier TtuB [16]. While two of these modified lysine residues (Lys226 and Lys229) are unique to TtuA, the third (Lys137) is conserved with P. horikoshii Ph0300 and is located within a disordered ‘hole-forming’ region near the disulfide bond forming cysteine residues proposed to function in catalysis of tRNA thiolation [17]. Thus, ubiquitin-like modification of TtuA at Lys137 is thought to alter enzyme activity [17]. TtuA Lys137 is not conserved with NcsA or Tuc1/Ncs6 homologs. However, the modified Lys204 of the Hfx. volcanii NcsA is located in close proximity to conserved active site cysteine and PP-motif residues based on 3D-homology modeling (Figure S2 in File S1). Thus, the ubiquitin-like modification of NcsA by SAMP2 may regulate its tRNA thiolase activity. Alternatively, poly-samp2ylation of NcsA may act as a signal for degradation by proteasomes and/or interaction with other proteins based on its location in a flexible loop and in analogy to the eukaryotic ubiquitin-proteasome system (UPS) (Figure 8B). Clearly further work is needed to determine the biological role of the observed polysampylation of NcsA.


Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

Model of NcsA activity and sampylation (panel A) as well as its association with protein partners (panel B).Panel A, NcsA is proposed to catalyze the formation of 2-thiouridine (s2U) at the wobble uridine position of tRNAs specific for lysine (tRNALysUUU), glutamate (tRNAGluUUC), and glutamine (tRNAGlnUUG) via an adenylated tRNA intermediate using thiocarboxylated SAMP2 as a source of activated sulfur. The E1-like UbaA adenylates the C-terminal α-carboxylate group of SAMP2. This modification readies the Ub-fold SAMP2 for either thiocarboxylation via an enzyme (cysteine desulfurase or rhodanese) catalyzed persulfide sulfur or protein modification via formation of a UbaA-SAMP2 thioester intermediate. Polymeric chains of SAMP2 are formed on an NcsA lysine residue via isopeptide linkages that are cleaved by HvJAMM1 protease. Whether additional factors are needed to provide specificity to the sampylation system is unclear, as E2 and E3 homologs are not predicted based on genome sequence. Panel B, NcsA is found isopeptide linked to SAMP2 and non-covalently associated with various proteins, as noted by dotted red lines. NcsA partners include the E1-like UbaA and Ub-fold SAMP2 of the tRNA thiolation and sampylation pathways. NcsA is also found associated with EF-1α that binds aminoacylated-tRNAs and mediates translation elongation, PAN-A/1 (an AAA+ATPase associated with energy-dependent proteolysis by proteasomes (20S core particles or CPs) and protein remodeling, and the β-CASP ribonuclease homolog of the aCPSF1 family thought to cleave mRNA and/or tRNA. RNAP, RNA polymerase.
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Related In: Results  -  Collection

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pone-0099104-g008: Model of NcsA activity and sampylation (panel A) as well as its association with protein partners (panel B).Panel A, NcsA is proposed to catalyze the formation of 2-thiouridine (s2U) at the wobble uridine position of tRNAs specific for lysine (tRNALysUUU), glutamate (tRNAGluUUC), and glutamine (tRNAGlnUUG) via an adenylated tRNA intermediate using thiocarboxylated SAMP2 as a source of activated sulfur. The E1-like UbaA adenylates the C-terminal α-carboxylate group of SAMP2. This modification readies the Ub-fold SAMP2 for either thiocarboxylation via an enzyme (cysteine desulfurase or rhodanese) catalyzed persulfide sulfur or protein modification via formation of a UbaA-SAMP2 thioester intermediate. Polymeric chains of SAMP2 are formed on an NcsA lysine residue via isopeptide linkages that are cleaved by HvJAMM1 protease. Whether additional factors are needed to provide specificity to the sampylation system is unclear, as E2 and E3 homologs are not predicted based on genome sequence. Panel B, NcsA is found isopeptide linked to SAMP2 and non-covalently associated with various proteins, as noted by dotted red lines. NcsA partners include the E1-like UbaA and Ub-fold SAMP2 of the tRNA thiolation and sampylation pathways. NcsA is also found associated with EF-1α that binds aminoacylated-tRNAs and mediates translation elongation, PAN-A/1 (an AAA+ATPase associated with energy-dependent proteolysis by proteasomes (20S core particles or CPs) and protein remodeling, and the β-CASP ribonuclease homolog of the aCPSF1 family thought to cleave mRNA and/or tRNA. RNAP, RNA polymerase.
Mentions: Based on this study, we propose a model in which NcsA is covalently modified at lysine residue(s) by isopeptide linked chains of the ubiquitin-fold SAMP2, through a mechanism that requires the ubiquitin-activating E1 enzyme homolog UbaA (Figure 8A). While polymeric chains of SAMP2/3 were previously detected in the Hfx. volcanii proteome by MS/MS [13], [25], it was unclear whether or not these chains were anchored to protein targets. The prokaryotic ubiquitin-like protein (Pup) of mycobacteria is not known to form polymeric chains [32]. Thus, NcsA is the first example of a target protein that is isopeptide-linked to polymeric chains of an ubiquitin-like protein modifier in prokaryotic cells. While the lysine residue (Lys204) of NscA that was found modified by poly-SAMP2 is not highly conserved among archaea and appears restricted to species of Haloferax and Natrialba, Lys204 is predicted to be in a unstructured region of NcsA (highlighted in Figure 1) that has undergone large amino acid insertions in some species (e.g., Haloarcula vallismortis GI:490650014). Interestingly, the tRNA thiolase of T. thermophilus, TtuA, was recently demonstrated to be isopeptide linked at multiple lysine residues (including Lys137, 226 and 229; highlighted in Figure 1) to the ubiquitin-like protein modifier TtuB [16]. While two of these modified lysine residues (Lys226 and Lys229) are unique to TtuA, the third (Lys137) is conserved with P. horikoshii Ph0300 and is located within a disordered ‘hole-forming’ region near the disulfide bond forming cysteine residues proposed to function in catalysis of tRNA thiolation [17]. Thus, ubiquitin-like modification of TtuA at Lys137 is thought to alter enzyme activity [17]. TtuA Lys137 is not conserved with NcsA or Tuc1/Ncs6 homologs. However, the modified Lys204 of the Hfx. volcanii NcsA is located in close proximity to conserved active site cysteine and PP-motif residues based on 3D-homology modeling (Figure S2 in File S1). Thus, the ubiquitin-like modification of NcsA by SAMP2 may regulate its tRNA thiolase activity. Alternatively, poly-samp2ylation of NcsA may act as a signal for degradation by proteasomes and/or interaction with other proteins based on its location in a flexible loop and in analogy to the eukaryotic ubiquitin-proteasome system (UPS) (Figure 8B). Clearly further work is needed to determine the biological role of the observed polysampylation of NcsA.

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Show MeSH