Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Nathanson, Lubov; Deutscher, Murray P.

doi:10.1074/jbc.c000385200

Cited by 70 publications

(76 citation statements)

References 29 publications

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“…S3B). Although it is formally possible that some nuclear-localized FTO that is weakly bound leaks into the cytosolic fraction during subcellular fractionation, it is also worth noting that a proportion of AARSs have been localized to the nucleus (24)(25)(26). Thus, the precise subcellular localization of the interaction between FTO and AARS remains an open question.…”

Section: Resultsmentioning

confidence: 99%

Role for the obesity-related FTO gene in the cellular sensing of amino acids

Gulati

Cheung

Antrobus

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

157

147

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SNPs in the first intron of FTO (fat mass and obesity associated) are strongly associated with human obesity. While it is not yet formally established that this effect is mediated through the actions of the FTO protein itself, loss of function mutations in FTO or its murine homologue Fto result in severe growth retardation, and mice globally overexpressing FTO are obese. The mechanisms through which FTO influences growth and body composition are unknown. We describe a role for FTO in the coupling of amino acid levels to mammalian target of rapamycin complex 1 signaling. These findings suggest that FTO may influence body composition through playing a role in cellular nutrient sensing.genetics | translation | tRNA synthetase | demethylase I n 2007, single nucleotide polymorphisms (SNPs) in the first intron of fat mass and obesity related (FTO) were found to be powerfully associated with body mass index and predisposing to childhood and adult obesity (1). Many subsequent studies, covering multiple populations of European, African, and Asian ancestries, across different age ranges, have confirmed the association of FTO with body mass index (reviewed in ref.2). It is clear from the weight of evidence that the major effect of SNPs in FTO is on increased energy intake, with reduction in satiety (3-8). To date, no conclusive link has been made between the risk alleles and expression or function of FTO. However, transgenic manipulation in mice supports the notion that FTO itself regulates body weight, with overexpression resulting in obesity (9), whereas Fto-null mice (10) and humans homozygous for a loss-of-function allele (11) display postnatal growth retardation and have high rates of early mortality.FTO is widely expressed across multiple tissues, although it is most highly expressed in the brain, especially in the hypothalamus, a region that plays a key role in the control of energy homeostasis (12). We have found that expression of FTO, specifically in the arcuate nucleus of the hypothalamus, is bidirectionally regulated as a function of nutritional status-decreasing following a 48-h fast and increasing after 10 wk of exposure to a high-fat diet-and that modulating FTO levels specifically in the arcuate nucleus can influence food intake (13). Carriers of the obesity predisposing allele are hyperphagic and show altered macronutrient preference.FTO shares sequence motifs with Fe(II)-and 2-oxoglutaratedependent oxygenases (12). In vitro, recombinant FTO is able to catalyze the Fe(II)-and 2-oxoglutarate-dependent demethylation of nucleic acids. It is more active at RNA than DNA (12,14) and is capable of demethylating N6-methyladenosine (15) (more prevalent on mRNAs) or 3-methyluracil (15) (more prevalent in tRNAs and ribosomal RNAs). To date, however, there has been no understanding of how FTO might functionally link to energy homeostasis, growth, or nutrient sensing. ResultsOne of the most striking features of mice lacking FTO (10), or humans homozygous for an enzymatically inactive mutated FTO (11), is severe growth ...

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Section: Resultsmentioning

confidence: 99%

Role for the obesity-related FTO gene in the cellular sensing of amino acids

Gulati

Cheung

Antrobus

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

157

147

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“…10B). The latter potentially damaging activity may be avoided in vivo because (i) mature tRNAs in both prokaryotes and eukaryotes appear to be almost fully aminoacylated except under conditions of nutritional deprivation (26,27) and (ii) eubacterial poly(A) polymerase preferentially tails readily denaturable mutant tRNAs, flagging them for subsequent degradation (28). Most revealingly, nucleotide specificity is compromised only with the tRNA-NCC substrate, not with the oligo(A) 15 primer (compare A and B in SI Fig.…”

Section: Resultsmentioning

confidence: 99%

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases

Cho

Verlinde

Weiner

2007

Proc. Natl. Acad. Sci. U.S.A.

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CCA-adding enzymes build and repair the 3 -terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC-and A-adding enzymes into UU-and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCAadding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2 hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 Å away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3 end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.nucleotidyltransferase ͉ tRNA T he CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase (NTR)] builds and repairs tRNA by adding the nucleotide sequence CCA to the 3Ј terminus of immature or damaged tRNA (1). Although this unusual RNA polymerase has no nucleic acid template, it constructs the CCA sequence one nucleotide at a time using CTP and ATP as substrates (1). All CCA-adding enzymes and poly(A) polymerases belong to the NTR superfamily, which can be divided into two distinct classes according to sequence motifs in the catalytic domain (2-6). The class I and class II CCA-adding enzymes exhibit little if any homology outside the NTR domain (6). Class I NTRs include archaeal CCA-adding enzymes, eukaryotic poly(A) polymerases, and probably eukaryotic terminal uridylyltransferases (7, 8); class II NTRs include eukaryotic and eubacterial CCAadding enzymes as well as eubacterial poly(A) polymerases (6).We found previously that a single NTR motif adds all three nucleotides (9, 10), that tRNA does not rotate or translocate along the enzyme during addition of C75 and A76 (11), and that a single active subunit in these dimeric or tetrameric enzymes can carry out all three steps of CCA addition (9). We therefore proposed that the growing 3Ј end of tRNA refolds progressively to reposition the new 3Ј hydroxyl identically relative to the single active site (10,12). This prediction was confirmed by cocrystal structures of the class I archaeal...

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“…For example, both EPRS and MRS are components of the MSC, and the rep domain, which is the region of EPRS targeted for sumoylation, is required for the formation of this complex (67). The functional significance of the MSC is unclear, although it may play a role in the trafficking of tRNA from the nucleus to the ribosome (47). Cellular stress and the resulting protein damage may lead to a need for increased protein synthesis, which in turn, would require increased levels of aminoacyl-tRNA at the ribosome.…”

Section: Sumo Deconjugation As a Means Tomentioning

confidence: 99%

“…In metazoans, about 9 of the ϳ20 aminoacyl-tRNA synthetases, including EPRS and MRS, associate with one another and additional factors to form an ϳ1.5 MDa protein complex termed the multiaminoacyl-tRNA synthetase complex (MSC) (46). While the MSC is primarily localized to the cytoplasm (the site of translation), a small fraction of the MSC is also found in the nucleus (47).…”

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confidence: 99%

Drosophila Ulp1, a Nuclear Pore-associated SUMO Protease, Prevents Accumulation of Cytoplasmic SUMO Conjugates

Smith

Bhaskar

Fernandez

et al. 2004

Journal of Biological Chemistry

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SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substratespecific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.The regulation of protein function by reversible post-translational modification is an effective means for regulating protein stability, activity, subcellular localization, and interaction specificity. One such modification involves the covalent attachment of the small ubiquitin-like modifier protein (SUMO) 1 (also called Smt3), to its target proteins via a series of enzymatic steps that closely resemble the reactions of the ubiquitylation pathway (1-3).Despite the sequence and structural homology between SUMO and ubiquitin, it is clear that sumoylation and ubiquitylation have distinct functional consequences. Ubiquitylation most frequently results in degradation of the substrate protein through targeting to the 26 S proteasome (4). The consequences of sumoylation are diverse and target-specific, with no evidence to suggest a role in promoting protein degradation. Conjugation of SUMO to RanGAP1 is required to target it to the nuclear pore complex (NPC), where it makes a stable interaction with the Ran-binding protein RanBP2 (5-7). Sumoylation of PML is essential for its recruitment to discrete intranuclear foci called PML oncogenic domains (PODs) (8 -11). For some sumoylation targets, including Smad4 and IB␣, this modification appears to increase protein stability by blocking ubiquitin-mediated degradation (12, 13). Finally, an increasing number of DNA binding transcription factors have been identified as substrates for sumoylation (14,15). Sumoylation of transcription factors can alter their activity in multiple ways. For example, in the ca...

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Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Cited by 70 publications

References 29 publications

Role for the obesity-related FTO gene in the cellular sensing of amino acids

Role for the obesity-related FTO gene in the cellular sensing of amino acids

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases

Drosophila Ulp1, a Nuclear Pore-associated SUMO Protease, Prevents Accumulation of Cytoplasmic SUMO Conjugates

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