Abstract
Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.
Highlights
The exact mechanism of isomerization is still not clear [4], the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins [10, 11] or ribonucleic acids (RNAs)-protein complexes [12]
liquid chromatography (LC)-mass spectrometry (MS) analysis of tRNATyr following treatment with RNase T1 and bacterial alkaline phosphatase (BAP) revealed a single response [40] for the triply charged oligonucleotide anion ACU[Q]UA[ms2i6A]A[⌿]CUG (m/z 1338.9, where U is the putative site of pseudouridine), which is a signature ion for E. coli tRNATyr [41]
cyclohexyl-NЈ--(4-methylmorpholinium)ethyl carbodiimide p-tosylate (CMCT)-treated RNA—LC-MS analysis of CMCT-derivatized T1 and BAP digest of E. coli tRNATyr I or II yielded very little signal for the underivatized oligonucleotide anion but produced a high abundance signal for m/z values that correspond to the digestion product with one and two carbodiimide units, respectively (Fig. 2, A and B), consistent with previous observations [28]
Summary
The exact mechanism of isomerization is still not clear [4], the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins (referred to as ⌿ synthases) [10, 11] or RNA-protein complexes (referred to as H/ACA box ribonucleoproteins) [12]. LC-MS analysis of tRNATyr following treatment with RNase T1 and bacterial alkaline phosphatase (BAP) revealed a single response [40] for the triply charged oligonucleotide anion ACU[Q]UA[ms2i6A]A[⌿]CUG (m/z 1338.9, where U is the putative site of pseudouridine), which is a signature ion for E. coli tRNATyr [41] (supplemental Fig. S1).
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