Introduction to tRNA Sequencing

Our understanding of gene expression has been transformed by high-throughput RNA sequencing (RNA-seq). RNA-seq methods often employed start with adaptor ligation and cDNA synthesis of biological RNA samples, then PCR amplification to build sequencing libraries1. Most cellular RNAs, such as mRNA, long ncRNA, miRNA, or fragments produced from rRNA, snRNA, and snoRNA, respond well to these simple methods. Although efforts have been made, standard sequencing methods have yet to be implemented effectively and quantitatively to tRNA, which is the only type of cellular RNA for which efforts have been made. The existence of various post-transcriptional modifications and its persistent and broad secondary structure, which interfere with cDNA synthesis and adaptor ligation, are significant barriers to tRNA sequencing. tRNAs are vital for cells, and their synthesis is tightly regulated by the cells. Evidence is mounting that tRNA expression and mutations are linked to a variety of illnesses, including neurological disorders and cancer development. Biological investigations of tRNA have been hampered by the absence of effective and quantitative tRNA-seq techniques.

Figure 1. Efficient and quantitative high-throughput tRNA sequencing. (Zheng, 2015)

Methods for Quantifying tRNA

Hybridization-based Approaches

The majority of earlier research relied on time-consuming hybridization-based methodologies such as array and northern blotting techniques to monitor tRNA levels. Although hybridization-based approaches can offer bulk quantitation for some tRNAs with the same anticodon, they are unable to identify some anticodon groups and isodecoders that differ by only one or a few nucleotides. Furthermore, arrays and northern blots do not reveal potential tRNA changes, which are thought to be critical for their function.

Next-generation RNA Sequencing

The demand for higher precision prompted the development of high-throughput sequencing methods capable of distinguishing tRNA genes at the isodecoder level. With the exception of tRNAs, next-generation RNA sequencing has changed modern molecular biology. Because of the aforementioned base changes and complex structures, tRNAs have historically been resistant to high-throughput sequencing. Many base changes cause Watson/Crick base pairing to be disrupted, and the inherent stem-loop structures prevent reverse transcriptase from synthesizing the initial strand (RT). To get around these problems, the few disclosed approaches use a variety of creative library preparation procedures. Zheng et al. presented the first procedure, particularly for sequencing tRNA, called DM-tRNA-seq. This procedure employs a more processive RT as well as AlkB, a pure prokaryotic demethylase, to remove a series of methyl groups from tRNA that induces RT stalling, resulting in a higher percentage of longer cDNA products.

Hydro-tRNA-Seq

This strategy improves coverage uniformity across a tRNA transcript by limiting tRNA fragmentation during library preparation to minimize changed bases. This fragmentation enables shorter tRNA fragment priming and cDNA synthesis.

YAMAT-Seq

The most recent approach, which uses a double-stranded adapter ligated to the 5′ and 3′ termini of mature tRNA, varies from Hydro-tRNA-seq and DM-tRNA-non-specific seq's adapter ligation and template switching phases, respectively. Due to the requirement for full-length cDNA, YAMAT-seq is unable to quantify a significant number of tRNAs, therefore highly structured and modified tRNA that RT cannot fully traverse are picked against. The lack of bias assessment and comprehensive cross-validation to test the accuracy of each technique is a key flaw in each of these protocols. Despite this, these technologies have significantly increased our understanding of tRNA biology and led to significant and crucial findings.

References:

1. Warren JM, Salinas-Giegé T, Hummel G, et al. Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification. RNA biology. 2021 Jan 2;18(1).
2. Pinkard O, McFarland S, Sweet T, Coller J. Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation. Nature communications. 2020 Aug 14;11(1).
3. Zheng G, Qin Y, Clark WC, et al. Efficient and quantitative high-throughput tRNA sequencing. Nature methods. 2015 Sep;12(9).