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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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Hfx. volcanii NcsA is required for thiolation of tRNALysUUU.Total RNA was isolated from H26 (wt, parent), ΔncsA (Δhvo_0580), and trans complemented ΔncsA strains, electrophoresed in a 12% urea polyacrylamide gel supplemented with 30 µg APM per ml, and hybridized to a probe complementary to tRNALysUUU by Northern blotting. For further details see Methods section. Thiolated tRNALysUUU migrates slower than non-thiolated tRNALysUUU as indicated.
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pone-0099104-g002: Hfx. volcanii NcsA is required for thiolation of tRNALysUUU.Total RNA was isolated from H26 (wt, parent), ΔncsA (Δhvo_0580), and trans complemented ΔncsA strains, electrophoresed in a 12% urea polyacrylamide gel supplemented with 30 µg APM per ml, and hybridized to a probe complementary to tRNALysUUU by Northern blotting. For further details see Methods section. Thiolated tRNALysUUU migrates slower than non-thiolated tRNALysUUU as indicated.

Mentions: We next used a genetic approach to investigate the role of NcsA in the thiolation of tRNA. Hfx. volcanii strains with a markerless deletion of the ncsA (hvo_0580) gene and its trans-complement (that expressed NscA with a C-terminal StrepII-tag, NcsA-StrepII) were generated from parent strain H26 and confirmed by Southern blotting, PCR and DNA sequence analyses (Figure S3 in File S1). Total RNA was purified from these strains and analyzed for thiolation of wobble uridine tRNA by use of acryloylaminophenylmercuric chloride (APM) gel electrophoresis coupled with Northern blotting using a probe specific for tRNALysUUU (Figure 2). The tRNALysUUU probe was chosen based on the presence of a uridine nucleoside in the wobble position of its anticodon specific for lysine tRNAs. By this experimental approach, a fraction of tRNALysUUU in the parent and trans-complement strains was found to be thio-modified (Figure 2, lanes 1 and 3). By contrast, only non-thiolated tRNALysUUU was detected in the ΔncsA mutant strain (Figure 2, lane 2). Taken together, these results revealed NcsA is required for the thiomodification of the wobble uridine of the tRNAUUU specific for lysine, similarly to what has been previously observed for the ubiquitin-fold SAMP2 and E1-like UbaA [12]. Thus, UbaA, SAMP and NcsA may function like the eukaryotic Uba4, Urm1 and Ncs6 in the thiol-modification of wobble uridine tRNAs (i.e., tRNALysUUU, tRNAGluUUC and tRNAGlnUUG).


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)

Hfx. volcanii NcsA is required for thiolation of tRNALysUUU.Total RNA was isolated from H26 (wt, parent), ΔncsA (Δhvo_0580), and trans complemented ΔncsA strains, electrophoresed in a 12% urea polyacrylamide gel supplemented with 30 µg APM per ml, and hybridized to a probe complementary to tRNALysUUU by Northern blotting. For further details see Methods section. Thiolated tRNALysUUU migrates slower than non-thiolated tRNALysUUU as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099104-g002: Hfx. volcanii NcsA is required for thiolation of tRNALysUUU.Total RNA was isolated from H26 (wt, parent), ΔncsA (Δhvo_0580), and trans complemented ΔncsA strains, electrophoresed in a 12% urea polyacrylamide gel supplemented with 30 µg APM per ml, and hybridized to a probe complementary to tRNALysUUU by Northern blotting. For further details see Methods section. Thiolated tRNALysUUU migrates slower than non-thiolated tRNALysUUU as indicated.
Mentions: We next used a genetic approach to investigate the role of NcsA in the thiolation of tRNA. Hfx. volcanii strains with a markerless deletion of the ncsA (hvo_0580) gene and its trans-complement (that expressed NscA with a C-terminal StrepII-tag, NcsA-StrepII) were generated from parent strain H26 and confirmed by Southern blotting, PCR and DNA sequence analyses (Figure S3 in File S1). Total RNA was purified from these strains and analyzed for thiolation of wobble uridine tRNA by use of acryloylaminophenylmercuric chloride (APM) gel electrophoresis coupled with Northern blotting using a probe specific for tRNALysUUU (Figure 2). The tRNALysUUU probe was chosen based on the presence of a uridine nucleoside in the wobble position of its anticodon specific for lysine tRNAs. By this experimental approach, a fraction of tRNALysUUU in the parent and trans-complement strains was found to be thio-modified (Figure 2, lanes 1 and 3). By contrast, only non-thiolated tRNALysUUU was detected in the ΔncsA mutant strain (Figure 2, lane 2). Taken together, these results revealed NcsA is required for the thiomodification of the wobble uridine of the tRNAUUU specific for lysine, similarly to what has been previously observed for the ubiquitin-fold SAMP2 and E1-like UbaA [12]. Thus, UbaA, SAMP and NcsA may function like the eukaryotic Uba4, Urm1 and Ncs6 in the thiol-modification of wobble uridine tRNAs (i.e., tRNALysUUU, tRNAGluUUC and tRNAGlnUUG).

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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