Involvement of protein kinase C in the control of tRNA modification with queuine in HeLa cells

Langgut, Werner; Reisser, Thomas

doi:10.1093/nar/23.13.2488

Cited by 10 publications

(7 citation statements)

References 19 publications

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“…Protein sequence homology of QTRT1 to the eubacterial TGT, along with co-purification studies, led to the proposal that the eukaryl TGT was a heterodimer of QTRT1 and ubiquitin-specific protease 14 (USP14). It was hypothesized that QTRT1 was responsible for the transglycosylase activity, whereas USP14 regulated the activity of QTRT1 in response to phosphorylation by protein kinase C (PKC) (Langgut and Reisser 1995;Morris et al 1995;Deshpande et al 1996). Other than the co-purification of QTRT1 and USP14 reported previously, there have not been any rigorous in vitro experiments to support this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Chen

Kelly

Stachura

et al. 2010

RNA

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The eukaryotic tRNA-guanine transglycosylase (TGT) has been reported to exist as a heterodimer, in contrast to the homodimeric eubacterial TGT. While ubiquitin-specific protease 14 (USP14) has been proposed to act as a regulatory subunit of the eukaryotic TGT, the mouse TGT has recently been shown to be a queuine tRNA-ribosyltransferase 1 (QTRT1, eubacterial TGT homolog) d queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1) heterodimer. We find that human QTRTD1 (hQTRTD1) co-purifies with polyhistidine-tagged human QTRT1 (ht-hQTRT1) via Ni 2+ affinity chromatography. Cross-linking experiments, mass spectrometry, and size exclusion chromatography results are consistent with the two proteins existing as a heterodimer. We have not been able to observe co-purification and/or association between hQTRT1 and USP14 when coexpressed in Escherichia coli. More importantly, under our experimental conditions, the transglycosylase activity of hQTRT1 is only observed when hQTRT1 and hQTRTD1 have been co-expressed and co-purified. Kinetic characterization of the human TGT (hQTRT1 d hQTRTD1) using human tRNA Tyr and guanine shows catalytic efficiency (k cat /K M ) similar to that of the E. coli TGT. Furthermore, site-directed mutagenesis confirms that the hQTRT1 subunit is responsible for the transglycosylase activity. Taken together, these results indicate that the human TGT is composed of a catalytic subunit, hQTRT1, and hQTRTD1, not USP14. hQTRTD1 has been implicated as the salvage enzyme that generates free queuine from QMP. Work is ongoing in our laboratory to confirm this activity.

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Section: Discussionmentioning

confidence: 99%

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Chen

Kelly

Stachura

et al. 2010

RNA

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“…Q-base and Ribosomal Frameshifting tRNAs within 72 hours (Langgut & Reisser, 1995). To this end, cells that had been passaged ten times in medium containing either HS or FCS were supplemented with free q-base at 300 nM and passaged a further ten times.…”

Section: Q-base Is Not Required For Efficient à1 Ribosomal Frameshiftmentioning

confidence: 99%

The Q-base of asparaginyl-tRNA is dispensable for efficient −1 ribosomal frameshifting in eukaryotes

Marczinke

Hagervall

Brierley

2000

Journal of Molecular Biology

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The frameshift signal of the avian coronavirus infectious bronchitis virus (IBV) contains two cis-acting signals essential for efficient frameshifting, a heptameric slippery sequence (UUUAAAC) and an RNA pseudoknot structure located downstream. The frameshift takes place at the slippery sequence with the two ribosome-bound tRNAs slipping back simultaneously by one nucleotide from the zero phase (U UUA AAC) to the -1 phase (UUU AAA). Asparaginyl-tRNA, which decodes the A-site codon AAC, has the modified base Q at the wobble position of the anticodon (5' QUU 3') and it has been speculated that Q may be required for frameshifting. To test this, we measured frameshifting in cos cells that had been passaged in growth medium containing calf serum or horse serum. Growth in horse serum, which contains no free queuine, eliminates Q from the cellular tRNA population upon repeated passage. Over ten cell passages, however, we found no significant difference in frameshift efficiency between the cell types, arguing against a role for Q in frameshifting. We confirmed that the cells cultured in horse serum were devoid of Q by purifying tRNAs and assessing their Q-content by tRNA transglycosylase assays and coupled HPLC-mass spectroscopy. Supplementation of the growth medium of cells grown either on horse serum or calf serum with free queuine had no effect on frameshifting either. These findings were recapitulated in an in vitro system using rabbit reticulocyte lysates that had been largely depleted of endogenous tRNAs and resupplemented with Q-free or Q-containing tRNA populations. Thus Q-base is not required for frameshifting at the IBV signal and some other explanation is required to account for the slipperiness of eukaryotic asparaginyl-tRNA.

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“…Activators and inhibitors of PKC modulate queuine uptake in cultured human fibroblasts. 24 Administrations of exogenous queuine to mouse improves Q-tRNA modification. [25][26][27] In the present study queuosine modification of transfer RNA is determined enzymatically by measuring the incorporation of radiolabelled guanine in transfer RNA.…”

Section: Introductionmentioning

confidence: 99%

Hypomodification of Transfer RNA in Cancer with Respect to Queuosine

Pathak¹,

Jaiswal²,

Vinayak³

2005

RNA Biology

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Involvement of protein kinase C in the control of tRNA modification with queuine in HeLa cells

Abstract: The eukaryotic tRNA:guanine transglycosylase (TGT) catalyses the base-for-base exchange of guanine for queuine (the q-base) a nutrition factor for eukary-

Cited by 10 publications

References 19 publications

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

The Q-base of asparaginyl-tRNA is dispensable for efficient −1 ribosomal frameshifting in eukaryotes

Hypomodification of Transfer RNA in Cancer with Respect to Queuosine

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