Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Transfer RNA (tRNA) expression and post-transcriptional modifications are crucial for biological processes.
• Sequencing tRNAs is difficult due to their structure and modifications, limiting high-throughput technologies.
• Flowering plants exhibit extensive tRNA post-transcriptional modifications.

Purpose of the Study:

• To develop and validate a modified RNA-seq protocol for sequencing full-length tRNAs and inferring modifications.
• To assess the utility of this method for studying tRNA expression and modifications in plants.
• To generate a tRNA modification index for *Arabidopsis thaliana*.

Main Methods:

• Modified RNA-seq protocol involving AlkB demethylation treatment and tRNA-specific adapter ligation.
• Sequencing of tRNAs from four flowering plant species.
• Northern blot analysis and droplet digital PCR for validation.

Main Results:

• The protocol successfully generated full-length tRNA sequences and inferred post-transcriptional modifications.
• A tRNA modification index for *Arabidopsis thaliana* was produced, revealing conserved and unique modification patterns.
• Despite improvements, tRNA-seq showed biased estimates of absolute expression levels due to reverse transcription.

Conclusions:

• The modified tRNA-seq protocol is promising for assessing relative tRNA expression and elucidating the functional roles of tRNA modifications.
• This method advances the study of tRNA biology, particularly in organisms with extensive modifications.
• Further optimization is needed to address biases in absolute expression quantification.