ABSTRACT
Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
Subject(s)
RNA, Transfer , Animals , Humans , Rats , Anticodon , Cell Line , Codon , Glycosylation , Nucleoside Q/chemistry , Nucleoside Q/genetics , Nucleoside Q/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Swine , Zebrafish/metabolism , Nucleic Acid ConformationABSTRACT
The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.
Subject(s)
Nucleotidyltransferases , RNA Caps , Vaccinia virus , Protein Biosynthesis , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/metabolism , Vaccinia virus/enzymology , Nucleotidyltransferases/chemistryABSTRACT
The 7-methylguanosine (m7G) cap structure, an essential epitranscriptomic mark at the 5' terminus of eukaryotic mRNAs, plays critical roles in mRNA stability, export, and translation. Following the cap structure, the first and second nucleotides at the 5' ends of mRNAs are frequently methylated to give more diverse modifications, especially in vertebrates. To understand the biological roles of the cap structures, precise analyses of the 5' terminal modifications are necessary. Here, we describe a detailed protocol for mass spectrometric analysis of 5' terminal fragments of mRNAs.
Subject(s)
Eukaryota , RNA Stability , Animals , Eukaryota/metabolism , Mass Spectrometry , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.
Subject(s)
Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Transfer/chemistry , Uridine/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Molecular Structure , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Uridine/genetics , Uridine/metabolismABSTRACT
N 6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N 6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N 6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N 6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.
