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Hypermethylated-capped selenoprotein mRNAs in mammals.

Wurth L, Gribling-Burrer AS, Verheggen C, Leichter M, Takeuchi A, Baudrey S, Martin F, Krol A, Bertrand E, Allmang C - Nucleic Acids Res. (2014)

Bottom Line: Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs.We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated.Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

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The cap of selenoprotein mRNAs is hypermethylated and poorly recognized by eIF4E. (A) Binding of recombinant GST-eIF4EK119A to HEK293 total RNA. GST-eIF4EK119A (or GST alone) was bound to glutathione beads and incubated with the extract. The percentage of mRNAs present in the bound and unbound fractions were determined separately by qRT-PCR and normalized to 100%. Asymmetric error bars represent the minimum and maximum observations for three independent biological replicates, reflecting intrinsic variability. SelR, GPx1, GPx4, SelM, SelW, SelT, SelO, TrxR1, Sel15, SelK, GPx3 and SelN are selenoprotein mRNAs. U3 snoRNA was used as a positive control, β-actin, HPRT and LDHA are housekeeping mRNAs used as negative controls. (B and C) Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-TMG serum (α-m3G). Bound RNA was analyzed by (B) RT-PCR or (C) qRT-PCR. (−) Control without antibodies. In: input 10%. The graph represents the percentage of mRNAs in IP compared with the input RNA. Error bars represent standard deviation of an average of three independent experiments. The horizontal line represents the level of housekeeping mRNA binding (1–2% in average). See also Supplementary Figure S1 for specificity controls. (D) Heatmap representation of mRNA binding in GST-eIF4E pull-down and TMG IP experiments. The binding scale is represented to the right, maximum binding values in each set of experiments are represented in red and minimal binding values in green. Heat maps were generated with the MeV software. The three classes of selenoprotein mRNAs are indicated.
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Figure 1: The cap of selenoprotein mRNAs is hypermethylated and poorly recognized by eIF4E. (A) Binding of recombinant GST-eIF4EK119A to HEK293 total RNA. GST-eIF4EK119A (or GST alone) was bound to glutathione beads and incubated with the extract. The percentage of mRNAs present in the bound and unbound fractions were determined separately by qRT-PCR and normalized to 100%. Asymmetric error bars represent the minimum and maximum observations for three independent biological replicates, reflecting intrinsic variability. SelR, GPx1, GPx4, SelM, SelW, SelT, SelO, TrxR1, Sel15, SelK, GPx3 and SelN are selenoprotein mRNAs. U3 snoRNA was used as a positive control, β-actin, HPRT and LDHA are housekeeping mRNAs used as negative controls. (B and C) Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-TMG serum (α-m3G). Bound RNA was analyzed by (B) RT-PCR or (C) qRT-PCR. (−) Control without antibodies. In: input 10%. The graph represents the percentage of mRNAs in IP compared with the input RNA. Error bars represent standard deviation of an average of three independent experiments. The horizontal line represents the level of housekeeping mRNA binding (1–2% in average). See also Supplementary Figure S1 for specificity controls. (D) Heatmap representation of mRNA binding in GST-eIF4E pull-down and TMG IP experiments. The binding scale is represented to the right, maximum binding values in each set of experiments are represented in red and minimal binding values in green. Heat maps were generated with the MeV software. The three classes of selenoprotein mRNAs are indicated.

Mentions: We performed glutathione-S-transferase (GST) pull-down experiments using total RNAs from HEK293FT cells and a high-affinity mutant of eIF4E, GST-eIF4EK119A (40). This mutant was developed for specific isolation of 5′ m7G-capped mRNAs and showed strict specificity but 10-fold higher affinity for the m7G cap. The RNA content of eIF4E bound and unbound fractions was determined by qRT-PCR analysis. For the detection of selenoprotein mRNAs, we used primers complementary to 12 out of the 25 selenoprotein mRNAs characterized in mammals (Figure 1A). β-actin, HPRT (hypoxanthine guanine phosphoribosyltransferase) and LDHA (lactate dehydrogenase A) mRNAs were used as the m7G-capped controls, U3 snoRNA and U2 snRNA for TMG-capped controls. Coherently, an average of 75% of the canonical β-actin, HPRT and LDHA mRNAs were recovered in the eIF4E bound fraction, whereas 74% of sn-, snoRNAs were found in the unbound fraction (Figure 1A). The results revealed that selenoprotein mRNAs showed differential binding patterns to eIF4E. The selenoprotein mRNAs of SelR, glutathione peroxidases 1 and 4 (GPx1, GPx4), SelM and SelW showed a distribution pattern similar to that of sn-, snoRNAs with only 20–35% of the mRNA recovered in the eIF4E bound fraction (Figure 1A). SelT, SelO, thioredoxin reductase 1 (TrxR1), Sel15, SelK selenoprotein mRNAs showed an intermediate pattern with over 50% of the mRNAs in the bound fraction (Figure 1A), whereas selenoprotein mRNAs comprising glutathione peroxidase 3 (GPx3) and selenoprotein N (SelN) were enriched up to 70% in the eIF4E bound fraction with patterns similar to non-selenoprotein mRNAs (Figure 1A).


Hypermethylated-capped selenoprotein mRNAs in mammals.

Wurth L, Gribling-Burrer AS, Verheggen C, Leichter M, Takeuchi A, Baudrey S, Martin F, Krol A, Bertrand E, Allmang C - Nucleic Acids Res. (2014)

The cap of selenoprotein mRNAs is hypermethylated and poorly recognized by eIF4E. (A) Binding of recombinant GST-eIF4EK119A to HEK293 total RNA. GST-eIF4EK119A (or GST alone) was bound to glutathione beads and incubated with the extract. The percentage of mRNAs present in the bound and unbound fractions were determined separately by qRT-PCR and normalized to 100%. Asymmetric error bars represent the minimum and maximum observations for three independent biological replicates, reflecting intrinsic variability. SelR, GPx1, GPx4, SelM, SelW, SelT, SelO, TrxR1, Sel15, SelK, GPx3 and SelN are selenoprotein mRNAs. U3 snoRNA was used as a positive control, β-actin, HPRT and LDHA are housekeeping mRNAs used as negative controls. (B and C) Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-TMG serum (α-m3G). Bound RNA was analyzed by (B) RT-PCR or (C) qRT-PCR. (−) Control without antibodies. In: input 10%. The graph represents the percentage of mRNAs in IP compared with the input RNA. Error bars represent standard deviation of an average of three independent experiments. The horizontal line represents the level of housekeeping mRNA binding (1–2% in average). See also Supplementary Figure S1 for specificity controls. (D) Heatmap representation of mRNA binding in GST-eIF4E pull-down and TMG IP experiments. The binding scale is represented to the right, maximum binding values in each set of experiments are represented in red and minimal binding values in green. Heat maps were generated with the MeV software. The three classes of selenoprotein mRNAs are indicated.
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Figure 1: The cap of selenoprotein mRNAs is hypermethylated and poorly recognized by eIF4E. (A) Binding of recombinant GST-eIF4EK119A to HEK293 total RNA. GST-eIF4EK119A (or GST alone) was bound to glutathione beads and incubated with the extract. The percentage of mRNAs present in the bound and unbound fractions were determined separately by qRT-PCR and normalized to 100%. Asymmetric error bars represent the minimum and maximum observations for three independent biological replicates, reflecting intrinsic variability. SelR, GPx1, GPx4, SelM, SelW, SelT, SelO, TrxR1, Sel15, SelK, GPx3 and SelN are selenoprotein mRNAs. U3 snoRNA was used as a positive control, β-actin, HPRT and LDHA are housekeeping mRNAs used as negative controls. (B and C) Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-TMG serum (α-m3G). Bound RNA was analyzed by (B) RT-PCR or (C) qRT-PCR. (−) Control without antibodies. In: input 10%. The graph represents the percentage of mRNAs in IP compared with the input RNA. Error bars represent standard deviation of an average of three independent experiments. The horizontal line represents the level of housekeeping mRNA binding (1–2% in average). See also Supplementary Figure S1 for specificity controls. (D) Heatmap representation of mRNA binding in GST-eIF4E pull-down and TMG IP experiments. The binding scale is represented to the right, maximum binding values in each set of experiments are represented in red and minimal binding values in green. Heat maps were generated with the MeV software. The three classes of selenoprotein mRNAs are indicated.
Mentions: We performed glutathione-S-transferase (GST) pull-down experiments using total RNAs from HEK293FT cells and a high-affinity mutant of eIF4E, GST-eIF4EK119A (40). This mutant was developed for specific isolation of 5′ m7G-capped mRNAs and showed strict specificity but 10-fold higher affinity for the m7G cap. The RNA content of eIF4E bound and unbound fractions was determined by qRT-PCR analysis. For the detection of selenoprotein mRNAs, we used primers complementary to 12 out of the 25 selenoprotein mRNAs characterized in mammals (Figure 1A). β-actin, HPRT (hypoxanthine guanine phosphoribosyltransferase) and LDHA (lactate dehydrogenase A) mRNAs were used as the m7G-capped controls, U3 snoRNA and U2 snRNA for TMG-capped controls. Coherently, an average of 75% of the canonical β-actin, HPRT and LDHA mRNAs were recovered in the eIF4E bound fraction, whereas 74% of sn-, snoRNAs were found in the unbound fraction (Figure 1A). The results revealed that selenoprotein mRNAs showed differential binding patterns to eIF4E. The selenoprotein mRNAs of SelR, glutathione peroxidases 1 and 4 (GPx1, GPx4), SelM and SelW showed a distribution pattern similar to that of sn-, snoRNAs with only 20–35% of the mRNA recovered in the eIF4E bound fraction (Figure 1A). SelT, SelO, thioredoxin reductase 1 (TrxR1), Sel15, SelK selenoprotein mRNAs showed an intermediate pattern with over 50% of the mRNAs in the bound fraction (Figure 1A), whereas selenoprotein mRNAs comprising glutathione peroxidase 3 (GPx3) and selenoprotein N (SelN) were enriched up to 70% in the eIF4E bound fraction with patterns similar to non-selenoprotein mRNAs (Figure 1A).

Bottom Line: Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs.We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated.Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.

Show MeSH
Related in: MedlinePlus