Hypermethylated-capped selenoprotein mRNAs in mammals.
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.
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
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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).
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.