Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications

Abstract

We report a direct method for sequencing tRNA Phe without cDNA by combining 2dimensional hydrophobic RNA end-labeling with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNA Phe was sequenced and the identity, location and abundance of all 11 base modifications were determined. Changes in ratios of wybutosine and its depurinated form under different conditions were quantified, pointing to the ability of our technology to determine dynamic changes of nucleotide modifications. Two truncated isoforms at 3´CCA tail of the tRNA Phe (75 nt CC, 80% and 74 nt C, 3%) were identified in addition to the 76 nt tRNA Phe with a full-length 3´CCA tail (17%). We also discovered a new isoform with A-G transitions at both the 44 and 45 positions in the tRNA Phe variable loop.

On the Track of the Missing tRNA Genes: A Source of Non-Canonical Functions?

“…An extraordinary volume of work has led to characterize the population of tRNAs in organisms that cover the entire phylogeny, both by analysis of tRNA molecules and genomic data (Chan and Lowe, 2009;Jühling et al, 2009;Lowe and Chan, 2016). Yet, it is interesting to notice that tRNAs, which have been the first sequenced nucleic acid molecule (Holley et al, 1965) and the first solved three-dimensional structure (Kim et al, 1974), still pose complex challenges centered on their structure and functions, despite the remarkable advances in sequencing techniques and structural studies of nucleic acids (Dittmar et al, 2006;Pang et al, 2014;Ferro and Ignatova, 2015;Zhang et al, 2015;Evans et al, 2017;Shigematsu et al, 2017;Kimura et al, 2020;Pinkard et al, 2020;Zhang et al, 2020). Old questions that focus on the structurefunction relationship and fidelity of the interactions and cellular processes in which these molecules participate are thus renewed.…”

MLC Seq: De novo sequencing of full-length tRNA isoforms by mass ladder complementation

“…Unlike MS-based RNA mapping methods, MS-based de novo sequencing methods rely on a complete set of MS ladders that are produced by exactly one random and unbiased cut on each RNA strand via controlled enzymatic or chemical degradation 7,10,15 . The ladder fragments carry nucleotide modifications from original parental RNAs, allowing identification, quantification and location of their nucleotide modifications 15,16 . However, each ladder must be perfect without any missing fragments in order to read all nucleotides in an RNA strand 15,16 .…”

“…The ladder fragments carry nucleotide modifications from original parental RNAs, allowing identification, quantification and location of their nucleotide modifications 15,16 . However, each ladder must be perfect without any missing fragments in order to read all nucleotides in an RNA strand 15,16 . As such, they can provide de novo sequence information themselves, without the need for prior sequence information and thus are independent of other methods like NGS.…”

“…We previously demonstrated the feasibility of using a MS laddering method for de novo sequencing of a mixture of 12 synthetic RNA strands with similar molar ratios, from which a perfect mass ladder was generated for each RNA strand without any missing ladder fragments 15 . This approach was also applied to sequencing of a yeast tRNA-Phe sample when coupled with a hydrophobic end-labeling strategy (HELS) 16 . However, only sequences of the major isoforms could be identified due to limited throughput from the sequencing algorithm which was tailored to report only the top-ranked sequences.…”

“…However, only sequences of the major isoforms could be identified due to limited throughput from the sequencing algorithm which was tailored to report only the top-ranked sequences. Moreover, due to the short read length (<~35 nt per run), this method required T1 digestion to decrease the input tRNA length (~80 nt) 16 .…”