Genome-wide analysis of the in vivo tRNA structurome reveals RNA structural and modification dynamics under heat stress

Abstract

RNA structure plays roles in myriad cellular events including transcription, translation, and RNA processing. Genome-wide analyses of RNA secondary structure in vivo by chemical probing have revealed critical structural features of mRNAs and long ncRNAs. Here, we examine the in vivo secondary structure of a small RNA class, tRNAs. Study of tRNA structure is challenging because tRNAs are heavily modified and strongly structured. We introduce “tRNA structure-seq,” a new workflow that accurately determines in vivo secondary structures of tRNA. The workflow combines dimethyl sulfate (DMS) probing, ultra-processive RT, and mutational profiling (MaP), which provides mutations opposite DMS and natural modifications thereby allowing multiple modifications to be identified in a single read. We applied tRNA structure-seq to E. coli under control and stress conditions. A leading folding algorithm predicts E. coli tRNA structures with only ∼80% average accuracy from sequence alone. Strikingly, tRNA structure-seq, by providing experimental restraints, improves structure prediction under in vivo conditions to ∼95% accuracy, with more than 14 tRNAs predicted completely correctly. tRNA structure-seq also quantifies the relative levels of tRNAs and their natural modifications at single nucleotide resolution, as validated by LC-MS/MS. Our application of tRNA structure-seq yields insights into tRNA structure in living cells, revealing that it is not immutable but has dynamics, with partial unfolding of secondary and tertiary tRNA structure under heat stress that is correlated with a loss of tRNA abundance. This method is applicable to other small RNAs, including those with natural modifications and highly structured regions.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase

“…1 A ). In the previous study, tRNA Structure-seq detected about 40% of E. coli natural tRNA modifications including 4-thiouridine (s 4 U), D, pseudouridine (Ψ), 2′- O -methylated nucleosides, various anticodon modifications, m 7 G, aminocarboxypropyluridine (acp 3 U), and 5-methyluridine (m 5 U) ( 40 ). In the original protocol, the RT reaction is performed using Marathon RTase in the presence of MnCl 2 .…”

“…This issue could be addressed by taking advantage of chemical agents such as sodium borohydride (NaBH 4 ) and rhodamine for dihydrouridine and 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) for pseudouridine ( 67 , 68 ) and coupling them to tRNA-MaP in the future. Recent studies show that the modification levels in tRNA are varied upon culture conditions and/or cellular stress in both prokaryotes and eukaryotes ( 12 , 40 , 69 ). The regulation of anticodon modification levels has a huge impact on the anticodon stem and loop structure and causes selective translation for stress response ( 69 , 70 ).…”

“…To tackle the obstacle, high-throughput tRNA-sequencing techniques have been developed for comprehensive tRNA analyses ( e.g. tRNA Modomics, tRNA abundance, and tRNA structurome) ( 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ). These techniques rely on a common basic strategy wherein tRNA sequences are converted into complementary DNA (cDNA) by reverse transcription (RT).…”

“…These techniques rely on a common basic strategy wherein tRNA sequences are converted into complementary DNA (cDNA) by reverse transcription (RT). During RT, some RNA modifications induce termination and/or nucleotide misincorporation into the cDNA which are recorded as RT stop counts and elevated mutation rates, respectively, in next-generation sequencing ( 40 ). These RT-derived signals allow one to predict the modification sites in tRNA.…”

“…Moreover, Yamagami et al. developed tRNA Structure-seq that predicts cellular tRNA secondary structures wherein both mutations in cDNA from natural tRNA modifications and chemical modifications from dimethyl sulfate that probes RNA secondary structure are simultaneously detected by Mutational Profiling (MaP) ( 40 ). However, despite the improvement of these tRNA sequencing techniques, to date none have been applied to functional analyses of tRNA modification enzyme.…”