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: The discovery of hypermethylated-capped selenoprotein mRNAs raises the fundamental question of their ability to be present and translated in the cytoplasm. Indeed, since the TMG cap is a part of the nuclear localization signal for snRNAs, it could also be envisaged that hypermethylation leads to sequestration of selenoprotein mRNAs in the nucleus. We thus performed subcellular fractionation of HEK293 cells (Figure 5A) followed by TMG-IP experiments and determined the percentage of each TMG-capped mRNA in the cytoplasm compared with the nucleus (Figure 5B). To assess the quality of the nuclear-cytoplasmic fractions, we have performed western blot analysis using antibodies directed against the transcription factor ZNF143 (a strictly nuclear protein (50)) and the cytoplasmic ribosomal protein rpS21 (Figure 5A). Results showed that globally selenoprotein mRNAs are more abundant in the cytoplasmic than the nuclear compartment; indeed 70–84% of TMG-capped selenoprotein mRNAs are found in the cytoplasm (Figure 5B). As expected, both U3 snoRNA and U6 snRNA were predominantly immunoprecipitated from the nuclear fraction. U6 was used as a control because it does not exit the nucleus during biogenesis; although it is not TMG-capped, it is always recovered in TMG-IPs because of its interaction with U4 snRNA (51). Non-selenoprotein mRNA controls were not recognized by the anti-TMG antibody. These results strongly suggest that hypermethylated-capped selenoprotein mRNAs are present in the cytoplasm, a prerequisite to their translation. To evaluate the ability of selenoprotein mRNAs to associate with actively translating ribosomes, we analyzed the polysome distribution of endogenous selenoprotein mRNAs. Cytoplasmic extracts of cycloheximide-treated HEK293 cells (blocking translation elongation) were fractionated on linear 7–47% sucrose gradients and the abundance of individual mRNAs in each fraction was measured by qRT-PCR (Figure 6A–C). The profiles revealed that all the endogenous selenoprotein mRNAs tested, except TrxR1, sedimented to fractions of lower molecular weight than non-selenoprotein mRNAs. The peak of selenoprotein mRNA population was found in fractions 10–26 in close proximity to the 80S monosomes (Figure 6B), whereas β-actin, HPRT and LDHA mRNAs sedimented in the heavier fractions 5–16 containing the polysomes (Figure 6C). As expected, U3 snoRNA was not found in polysomes (Figure 6C). These results indicate that fewer ribosomes are loaded on selenoprotein mRNAs, consistent with elongation pausing at the UGA Sec codon as previously reported for GPx4 (52). TrxR1 selenoprotein mRNA is the only mRNA found in heavier polysome fractions (see Figure 6B). This correlates with the fact that TrxR1 is the sole mRNA tested for which the UGA Sec codon is at the antepenultimate position before the stop codon; in all the other selenoprotein mRNAs of our test panel, the UGA Sec codon resides between 39 (SelW) and 285 nucleotides (SelR) downstream of the start codon. Worth of note, the proportion of free versus ribosome bound mRNAs is higher for selenoprotein than non-selenoprotein mRNAs. We next tested whether selenoprotein mRNAs present in polysomes bear hypermethylated caps. TMG-IP experiments were performed on pooled fractions 6-26 (polysomes) or 28-41 (RNP) that contain free or non-polysome associated RNAs. We reasonably considered that the two pools contained 100% of the RNAs. Immunoprecipitation yields dropped importantly after fractionation of the RNAs on sucrose gradients; nevertheless, the results clearly showed the presence of the hypermethylated selenoprotein mRNAs in the polysome pool (Figure 6D). Indeed, between 50% (SelT) and 80% (SelR) of the recovered TMG-capped mRNAs was present in the polysome fraction, the rest of the TMG-capped selenoprotein mRNAs being in the RNP fractions (Figure 6D). As expected, U3 snoRNA and U2 snRNA were predominantly recovered from the RNP pool (Figure 6D). The non-selenoprotein mRNA controls were not recognized by anti-TMG antibodies (see also Figure 1C). When RNA fractionation was performed under low magnesium concentration leading to ribosome dissociation into subunits (Figure 6A), the signal of selenoprotein mRNA TMG-IP was shifted to the RNP fractions (Figure 6E). Altogether these results show that hypermethylated selenoprotein mRNAs are found in polysomes and are therefore translated.
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.