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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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NcsA forms a complex with an archaeal CPSF1 homolog.A) Cell lysate and B) StrepII purified fractions of an ΔncsA mutant expressing NcsA-StrepII and/or Flag-aCPSF were probed with α-Flag and α-StrepII antibodies as indicated. C) Coomassie blue (CB) stain of StrepII-purified fractions as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 50 ml cultures. See Methods section for details.
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pone-0099104-g007: NcsA forms a complex with an archaeal CPSF1 homolog.A) Cell lysate and B) StrepII purified fractions of an ΔncsA mutant expressing NcsA-StrepII and/or Flag-aCPSF were probed with α-Flag and α-StrepII antibodies as indicated. C) Coomassie blue (CB) stain of StrepII-purified fractions as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 50 ml cultures. See Methods section for details.

Mentions: To further investigate the physical association of NcsA and aCPSF1 detected by our MS-analysis, the Hfx. volcanii aCPSF1 homolog (HVO_0874) was N-terminally Flag-tagged and expressed with and without NcsA-StrepII in a ΔncsA mutant background. aCPSF and NcsA synthesis were confirmed by anti-Flag and -StrepII IB of cell lysate, respectively (Figure 7A). Interestingly, when aCPSF1 was expressed alone, a ∼100 kDa protein was readily detected in cell lysate by α-Flag immunoblot that migrated similarly (although larger) than the Flag-aCPSF1 of 73.2 kDa theoretical mass based on genome sequence (Figure 7A, lane 3). By contrast, multiple aCPSF1 bands of 50–200 kDa were detected when NcsA was included in the expression strain (Figure 7A, lane 4), suggesting that, when NcsA is present in the cell, the aCPSF1 undergoes covalent modifications that alter its molecular mass with the lower molecular masses most likely due to proteolytic cleavage.


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)

NcsA forms a complex with an archaeal CPSF1 homolog.A) Cell lysate and B) StrepII purified fractions of an ΔncsA mutant expressing NcsA-StrepII and/or Flag-aCPSF were probed with α-Flag and α-StrepII antibodies as indicated. C) Coomassie blue (CB) stain of StrepII-purified fractions as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 50 ml cultures. See Methods section for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048286&req=5

pone-0099104-g007: NcsA forms a complex with an archaeal CPSF1 homolog.A) Cell lysate and B) StrepII purified fractions of an ΔncsA mutant expressing NcsA-StrepII and/or Flag-aCPSF were probed with α-Flag and α-StrepII antibodies as indicated. C) Coomassie blue (CB) stain of StrepII-purified fractions as indicated. Molecular weight markers are indicated to the left of each blot. Pull down assays were from 50 ml cultures. See Methods section for details.
Mentions: To further investigate the physical association of NcsA and aCPSF1 detected by our MS-analysis, the Hfx. volcanii aCPSF1 homolog (HVO_0874) was N-terminally Flag-tagged and expressed with and without NcsA-StrepII in a ΔncsA mutant background. aCPSF and NcsA synthesis were confirmed by anti-Flag and -StrepII IB of cell lysate, respectively (Figure 7A). Interestingly, when aCPSF1 was expressed alone, a ∼100 kDa protein was readily detected in cell lysate by α-Flag immunoblot that migrated similarly (although larger) than the Flag-aCPSF1 of 73.2 kDa theoretical mass based on genome sequence (Figure 7A, lane 3). By contrast, multiple aCPSF1 bands of 50–200 kDa were detected when NcsA was included in the expression strain (Figure 7A, lane 4), suggesting that, when NcsA is present in the cell, the aCPSF1 undergoes covalent modifications that alter its molecular mass with the lower molecular masses most likely due to proteolytic cleavage.

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