Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein

Sikorska, Marianna; Lm, Brewer; Youdale, T.; Richards, Robert I.; Jf, Whitfield; Houghten, R A; Pr, Walker

doi:10.1139/o90-130

Cited by 16 publications

(4 citation statements)

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“…In this study, RRM1 was detected to be located near the nucleus in SB cells and GS cells, suggesting that RRM1 may play a role in nuclear membrane. In another study, it demonstrates that RRM1 localizes to the nuclear membrane in the rat liver cell, which is consistent with our results (Sikorska et al 1990). However, in other reports, different cell types including non-small-cell lung cancer (NSCLC) cell lines (Zheng et al 2007) and in human lung fibroblasts cells (Engström and Rozell 1988), RRM1 is expressed in the nuclear and cytoplasmic regions, respectively, suggesting that localization of RRM1 depends on cell type and cellular state.…”

Section: Rrm1 Expression Analysis and Subcellular Localization In Sb Cellssupporting

confidence: 92%

Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing

2021

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Viral nervous necrosis (VNN) disease caused by the nervous necrosis virus (NNV) is a major disease, leading to a huge economic loss in aquaculture. Previous GWAS and QTL mapping have identified a major QTL for NNV resistance in linkage group 20 in Asian seabass. However, no causative gene for NNV resistance has been identified. In this study, RNA-seq from brains of Asian seabass fingerlings challenged with NNV at four time points (5, 10, 15 and 20 days post-challenge) identified 1228, 245, 189 and 134 DEGs, respectively. Eight DEGs, including rrm1, were located in the major QTL for NNV resistance. An association study in 445 survived and 608 dead fingerlings after NNV challenge revealed that the SNP in rrm1 were significantly associated with NNV resistance. Therefore, rrm1 was selected for functional analysis, as a candidate gene for NNV resistance. The expression of rrm1 was significantly increased in the gill, liver, spleen and muscle, and was suppressed in the brain, gut and skin after NNV challenge. The rrm1 protein was localized in the nuclear membrane. Overexpression of rrm1 significantly decreased viral RNA and titer in NNV-infected Asian seabass cells, whereas knock-down of rrm1 significantly increased viral RNA and titer in NNV-infected Asian seabass cells. The rrm1 knockout heterozygous zebrafish was more susceptible to NNV infection. Our study suggests that rrm1 is one of the causative genes for NNV resistance and the SNP in the gene may be applied for accelerating genetic improvement for NNV resistance.

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Section: Rrm1 Expression Analysis and Subcellular Localization In Sb Cellssupporting

confidence: 92%

Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing

2021

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“…Immunocytochemical studies showed that the R1 and R2 subunits of the mammalian RNR enzyme are both localized in the cytoplasm (11) and in some cell types appear to be concentrated near the nuclear region (28). The R2 of the herpes simplex virus type 1 RNR enzyme is concentrated in discrete cytoplasmic foci close to the nucleus, whereas R1 exhibits a more diffuse cytoplasmic localization (4).…”

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confidence: 99%

Identification of RNR4, Encoding a Second Essential Small Subunit of Ribonucleotide Reductase in Saccharomyces cerevisiae

Huang

Elledge

1997

Molecular and Cellular Biology

172

201

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Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an ␣ 2 ␤ 2 tetramer consisting of two large and two small subunits. RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae. We have cloned a second essential gene encoding a homologous small subunit, RNR4. RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced. The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes. RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts. Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes. Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.Ribonucleotide reductases (RNRs) catalyze the reduction of all four ribonucleoside diphosphates into deoxyribonucleoside diphosphates and play a central role in controlling the levels of cellular deoxynucleoside triphosphates (dNTPs), which are essential for high-fidelity DNA replication and DNA repair processes. Three classes of RNR enzymes have been characterized; they have unrelated protein structures, but all contain an organic free radical that is crucial for the catalytic activity, and all are allosterically regulated (for reviews, see references 23 and 31).Class I enzymes are the most commonly found and best studied. They exist in all eukaryotes and in most prokaryotes, with the aerobic Escherichia coli RNR as the prototype. The functional class I enzyme complex is a heterotetramer (␣ 2 ␤ 2 ) between two nonidentical homodimers (20), the large subunit (␣ or R1) and the small subunit (␤ or R2), each of which is inactive by itself. Each monomer of the R1 dimer (two 90-kDa monomers) contains one substrate-binding site with redoxactive thiols and two separate allosterically regulated binding sites for dNTPs. One site binds to ATP and dATP and controls the overall enzyme activity to ensure the proper balance between the level of NTPs for RNA synthesis and that of dNTPs for DNA synthesis; the other site controls the substrate specificity to maintain balanced dNTP pools that are necessary for high-fidelity DNA replication. Each monomer of the R2 dimer (two 45-kDa monomers) contains a tyrosyl free radical that is essential for catalytic activity. This tyrosyl free radical is generated and maintained by an oxygen-linked binuclear ferric iron center buried inside the protein (22). The chemotherapeutic agent hydroxyurea (HU) scavenges this tyrosyl radical and thereby inactivates RNR activity (2). The flexible carboxylterminal tail of the R2 protein is essential for binding to...

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“…Although RNR activity has been reported to be present in the outer nuclear membrane, most RNR activity (75%) is present in the cytosol. Furthermore, the changes in the activity in the two fractions are similar during the DNA synthesis phase [37]. To determine the effect of OA on the induction of RNR activity, rats were subjected to two-thirds PH, at which time, in some animals, a 300-mg tablet of OA was implanted i.p.…”

Section: Resultsmentioning

confidence: 99%

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

et al. 1999

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Our earlier studies have shown that in normal hepatocytes, orotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and after two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA induces an imbalance in nucleotide pools (specifically, an increase in uridine nucleotides, including deoxyuridine nucleotides, and a decrease in adenosine nucleotides, including ATP) and creation of this imbalance is crucial for the mitoinhibitory effects of OA, we hypothesized that ribonucleoside diphosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the inhibition of DNA synthesis by OA. To test this hypothesis, we subjected male Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the presence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneally at the time of two-thirds PH). The rats were killed at different times later, and their livers were processed for analysis of levels of RNR enzyme activity, protein, and mRNA transcripts. The results obtained indicated that treatment with OA resulted in a near-100% inhibition of RNR induced by two-thirds PH in rat liver, as monitored by enzyme activity and protein level. Furthermore, this inhibition was paralleled by a decrease in the mRNA transcripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indicated that this decrease in the levels of mRNA transcripts could not be attributed to an effect on transcription. However, administration of OA 20 h after two-thirds PH, when RNR mRNA transcripts were maximally induced, resulted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by two-thirds PH, at the levels of enzyme activity, protein, and mRNA transcripts, and the decreased levels of mRNA transcripts appeared to be due to increased degradation of the transcripts.

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Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein

Cited by 16 publications

References 1 publication

Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing

Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing

Identification of RNR4, Encoding a Second Essential Small Subunit of Ribonucleotide Reductase in Saccharomyces cerevisiae

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo

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