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RNA sequencing and proteogenomics reveal the importance of leaderless mRNAs in the radiation-tolerant bacterium Deinococcus deserti.

de Groot A, Roche D, Fernandez B, Ludanyi M, Cruveiller S, Pignol D, Vallenet D, Armengaud J, Blanchard L - Genome Biol Evol (2014)

Bottom Line: Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides.Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed.On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: CEA, DSV, IBEB, Lab Bioénergétique Cellulaire, Saint-Paul-lez-Durance, France.

ABSTRACT
Deinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing (RNA-seq) was performed to explore the specificities of its transcriptome. Strikingly, for 1,174 (60%) mRNAs, the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and nonannotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed reannotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed. On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.

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The 5′-end of cysS mRNA encodes a ten-amino-acid-long leader peptide with two cysteine residues. cysS codes for cysteinyl-tRNA synthetase. Start codons, -10 motif (upstream of TSS), SD sequence (upstream of cysS start codon), and 10 aa peptide are boxed. Panel (B) is a zoom at the TSS for cysS, which is also the translation start of the predicted leader peptide. Panel (C) is a zoom at the translation start of cysS. Treatment (+) or not (–) of RNA with TEX is indicated.
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evu069-F6: The 5′-end of cysS mRNA encodes a ten-amino-acid-long leader peptide with two cysteine residues. cysS codes for cysteinyl-tRNA synthetase. Start codons, -10 motif (upstream of TSS), SD sequence (upstream of cysS start codon), and 10 aa peptide are boxed. Panel (B) is a zoom at the TSS for cysS, which is also the translation start of the predicted leader peptide. Panel (C) is a zoom at the translation start of cysS. Treatment (+) or not (–) of RNA with TEX is indicated.

Mentions: Unexpectedly, for numerous genes, the gTSS was found at exactly the first nucleotide of the translation initiation codon ATG or GTG, or within a few nt upstream of the start codon (fig. 2; supplementary table S3, Supplementary Material online). Figure 3 shows an example of such a leaderless gene, with the TSS at the GTG start codon of irrE (Deide_03030), a gene essential for radiation tolerance (Vujicic-Zagar et al. 2009). Leaderless mRNAs lack an SD sequence or other regulatory structures that are generally present in the 5′-untranslated region (5′-UTR) of leadered mRNAs. Previous studies have indicated that the upper limit for a 5′-UTR to allow usage of the leaderless translation pathway is around 5 nt, with most efficient translation when the start codon is directly at the 5′-terminus (Hering et al. 2009; Krishnan et al. 2010). Therefore, mRNAs with a 5′-UTR of less than 6 nt were classified as leaderless. For the total genome, 1,174 (60%) of the 1,958 identified gTSSs correspond to leaderless mRNA (5′-UTR < 6 nt), with 916 (47%) of these gTSSs located exactly at the first nucleotide of the translation initiation codon (5′-UTR = 0 nt) (table 1). An even higher percentage of leaderless mRNA was found for the main chromosome (table 1). Using MEME (Bailey and Elkan 1994), a conserved motif resembling the -10 box TATAAT was found directly upstream of 94% of the TSSs (fig. 2; supplementary table S5, Supplementary Material online; see also figs. 1, 3, 5, and 6). A widely conserved -35 motif was not detected using the same approach. While the -10 motif was thus found directly upstream of the start codon of leaderless genes, the SD motif shown in figure 2 was found upstream of the start codon in 62% of the leadered mRNAs (supplementary table S6, Supplementary Material online; see also figures 1 and 6), which supports the identification of two types of mRNAs, that is, leaderless and leadered. The remaining 38% of the leadered mRNAs may contain a less conserved SD motif or might be translated in an SD-independent manner.Fig. 2.—


RNA sequencing and proteogenomics reveal the importance of leaderless mRNAs in the radiation-tolerant bacterium Deinococcus deserti.

de Groot A, Roche D, Fernandez B, Ludanyi M, Cruveiller S, Pignol D, Vallenet D, Armengaud J, Blanchard L - Genome Biol Evol (2014)

The 5′-end of cysS mRNA encodes a ten-amino-acid-long leader peptide with two cysteine residues. cysS codes for cysteinyl-tRNA synthetase. Start codons, -10 motif (upstream of TSS), SD sequence (upstream of cysS start codon), and 10 aa peptide are boxed. Panel (B) is a zoom at the TSS for cysS, which is also the translation start of the predicted leader peptide. Panel (C) is a zoom at the translation start of cysS. Treatment (+) or not (–) of RNA with TEX is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007540&req=5

evu069-F6: The 5′-end of cysS mRNA encodes a ten-amino-acid-long leader peptide with two cysteine residues. cysS codes for cysteinyl-tRNA synthetase. Start codons, -10 motif (upstream of TSS), SD sequence (upstream of cysS start codon), and 10 aa peptide are boxed. Panel (B) is a zoom at the TSS for cysS, which is also the translation start of the predicted leader peptide. Panel (C) is a zoom at the translation start of cysS. Treatment (+) or not (–) of RNA with TEX is indicated.
Mentions: Unexpectedly, for numerous genes, the gTSS was found at exactly the first nucleotide of the translation initiation codon ATG or GTG, or within a few nt upstream of the start codon (fig. 2; supplementary table S3, Supplementary Material online). Figure 3 shows an example of such a leaderless gene, with the TSS at the GTG start codon of irrE (Deide_03030), a gene essential for radiation tolerance (Vujicic-Zagar et al. 2009). Leaderless mRNAs lack an SD sequence or other regulatory structures that are generally present in the 5′-untranslated region (5′-UTR) of leadered mRNAs. Previous studies have indicated that the upper limit for a 5′-UTR to allow usage of the leaderless translation pathway is around 5 nt, with most efficient translation when the start codon is directly at the 5′-terminus (Hering et al. 2009; Krishnan et al. 2010). Therefore, mRNAs with a 5′-UTR of less than 6 nt were classified as leaderless. For the total genome, 1,174 (60%) of the 1,958 identified gTSSs correspond to leaderless mRNA (5′-UTR < 6 nt), with 916 (47%) of these gTSSs located exactly at the first nucleotide of the translation initiation codon (5′-UTR = 0 nt) (table 1). An even higher percentage of leaderless mRNA was found for the main chromosome (table 1). Using MEME (Bailey and Elkan 1994), a conserved motif resembling the -10 box TATAAT was found directly upstream of 94% of the TSSs (fig. 2; supplementary table S5, Supplementary Material online; see also figs. 1, 3, 5, and 6). A widely conserved -35 motif was not detected using the same approach. While the -10 motif was thus found directly upstream of the start codon of leaderless genes, the SD motif shown in figure 2 was found upstream of the start codon in 62% of the leadered mRNAs (supplementary table S6, Supplementary Material online; see also figures 1 and 6), which supports the identification of two types of mRNAs, that is, leaderless and leadered. The remaining 38% of the leadered mRNAs may contain a less conserved SD motif or might be translated in an SD-independent manner.Fig. 2.—

Bottom Line: Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides.Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed.On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: CEA, DSV, IBEB, Lab Bioénergétique Cellulaire, Saint-Paul-lez-Durance, France.

ABSTRACT
Deinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing (RNA-seq) was performed to explore the specificities of its transcriptome. Strikingly, for 1,174 (60%) mRNAs, the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and nonannotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed reannotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed. On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.

Show MeSH