Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Abstract

Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.

A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine

“…To ensure Q modification in S. pombe , the growth medium was supplemented with queuine, since this type of yeast, like other eukaryotes, lacks the enzymes of the queuosine biosynthetic pathway and therefore relies on external sources for Q ( 30 , 46 ). Feeding yeast cells with queuine induces Q modification of the four tRNAs with a GUN anticodon to >95% ( 45 ). Total RNA was isolated, and tRNA Asp was reverse transcribed with RT-KTq I614Y as above.…”

“…Following precipitation, total RNA was washed with 70% ethanol and dissolved in DEPC-treated water. Verification of Q modification in S. pombe and S. flexneri was performed by separating RNAs in 10% polyacrylamide gels [acrylamide/bisacrylamide (19:1), urea 8 M] supplemented with 5 mg/ml 3-(acrylamido)phenylboronic acid (APB) as described previously ( 44 ) followed by northern blotting as previously described ( 45 ).…”

“…The pCDF-Duet1 vector coexpressing the human TGT (hTGT) heterodimer QTRT1 and QTRT2 with a cleavable N-terminal 6xHis tag to QTRT1 was kindly provided by Prof. Dr Ralf Ficner (GZMB, Göttingen). Expression and purification of hTGT and Q modification of in vitro -transcribed tRNAs were carried out as previously described ( 45 ). Q modification was verified by separating RNAs in polyacrylamide gels containing APB (see above).…”

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications

“…Another important technology is mass spectrometry of nucleic acids, in which German researchers are clearly among the world leaders. Here, too, there have been strong and widely visible publications from SPP1784, especially in method development, e.g., by labeling with stable isotopes. − …”

“…47−51 Insights from molecular recognition have also been used for targeted manipulation of DNA, again specifically related to the above-mentioned cytidine derivatives. 57,58 In the chemical biology of cofactors, one focus was on SAM analogs, 14,28,59 another was on canonical and noncanonical cap structures and the structural elucidation of related enzymes. 60−62 Structural elucidation by synthesis of complicated hypermodifications from tRNAs 63,64 was in the spirit of a general revival of the functional analysis of such modifications.…”