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

Abstract

tRNAs can exist in distinct isoforms because of different chemical modifications, which confounds attempts to accurately sequence individual tRNA species using next generation sequencing approaches or to quantify different RNA modifications at specific sites on a tRNA strand. Herein, we develop a mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), allowing for direct and simultaneous sequencing of full-length tRNA molecules, including those with low abundance. MLC-seq is achieved by improved instrumentation, and advanced algorithms that identify each tRNA species and related isoforms in an RNA mixture, and assemble full MS ladders from partial ladders with missing ladder components. Using MLC-Seq, we successfully obtained the sequence of tRNA-Phe from yeast and tRNA-Glu from mouse hepatocytes, and simultaneously revealed new tRNA isoforms derived from nucleotide modifications. Importantly, MLC-Seq pinpointed the location and stoichiometry changes of RNA modifications in tRNA-Glu upon the treatment of dealkylated enzyme AlkB, which confirmed its known enzymatic activity and suggested previously unidentified effects in RNA editing.

Adaptor Molecules Epitranscriptome Reprograms Bacterial Pathogenicity

“…Unlike other established MSbased RNA mapping methods, a novel and NGS-independent methodology, termed mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), has been very recently described, relying on the construction of high quality tRNA ladders and the use of advanced algorithms. Without the requirement of prior NGS data, MLC-seq allows for the direct sequence of full length tRNA molecules and the simultaneous identification of different tRNA species and the contained modifications, overcoming the obstacles incorporated with the cDNA synthesis during the standard NGS-based methodologies [91].…”

Small RNAs: An expanding world with therapeutic promises

“…This awaits the development of new approaches that can simultaneously profile multiple RNA modifications. To explore the sncRNA kingdom further, combining NGS methods with mass spectrometry, such as MS ladder complementation sequencing (MLC-seq) [163] , as well as direct sequencing, may provide alternative solutions [ 33 , 164 ]. Additionally, profiling of novel sncRNAs and their modifications in rare samples, such as preimplantation embryos, remains mostly unachievable, presenting obstacles to further understanding in areas such as epigenetic inheritance.…”