mim-tRNA sequencing

tRNA, tiRNA and tRFs

Precise quantitative tRNA profiling of tRNA expression and modification

Transfer RNAs (tRNAs) are short, abundant transcripts needed for translating messenger RNA into protein. As such, the composition of cellular tRNA pools impact proteome dynamics and integrity. tRNA expression is dynamically regulated in different tissues and during development, and defective tRNA biogenesis is linked to neurological disorders and cancer.

What makes tRNA sequencing challenging?

Classic next generation sequencing has been challenged in measuring cellular tRNA abundance, due to pervasive blocks of cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes.

The omiics approach for tRNA analysis:

At omiics, we have developed a specialized protocol, inspired by the mim-tRNA approach.

Mim-tRNA sequencing overcome the classic issues described above and hans been used to uncover a dramatic heterogenety of tRNA pools between human cell lines and also showed there to be a interdependence of modifications within an individual tRNA transcript.

High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. A. Behrens et al.

Molecular Cell, Volume 81, Issue 8, 15 April 2021, Pages 1802-1815.e7

Results of the analysis

COVERAGE PLOT

A coverage plot is a type of graph that shows how much sequencing data (reads) you have at each position along each tRNA molecule.

As the polymerase moves along the sequence, especially for heavily modified regions, there is a risk of polymerase run-off/dissociation from the template. Also, if the template is fragmented, e.g. for tRFs, you will only get the fragment region sequenced.

DIFFERENTIAL EXPRESSION

Differential expression is a comparative method that provides relative values for the change in individual RNA molecules between two or more groups of samples (e.g. "treatment" and "control"). See the plots here

Because the result is a list of the changes in all RNAs between your two groups, it is a strong tool for exploratory studies with no pre-defined molecule of interest

CHARGING QUANTITATION

Aminoacylation, also known as tRNA charging, refers to whether a tRNA molecule is "loaded" with its corresponding amino acid.

By applying Omiics’ optimized protocol for mim-tRNA sequencing, you can determine the charging ratiofor each individual tRNA — expressed as the percentage of tRNAs that are aminoacylated (charged) versus those that are uncharged.

MODIFICATION PROFILING

The method identify and compare specific modifications at single-nucleotide resolution.

The most commonly profiled modifications include:

m¹A (1-methyladenosine)
m²,²G (N2,N2-dimethylguanosine)
m⁵C (5-methylcytidine)
m⁷G (7-methylguanosine)
Ψ (pseudouridine)
D (dihydrouridine)
Inosine (I)

A customer from a venture capital–backed spinout from a university, focused on developing a novel drug candidate.

Early studies had shown promising phenotypic effects in cell culture, indicating potential therapeutic value. However, the team lacked insight into the underlying molecular mechanisms responsible for these effects. This is an essential requirement both for attracting further investment in the short term and for meeting regulatory demands in the long term.

Case: Drug-candidate affects tRNA abundance and modification

Treated

Untreated

Challenge:

To assess the effect of a drug candidate in vitro on cellular mechanisms, to directly link drug treatment

Solution:

Omiics applied both established and novel methods to treated and untreated cell culture samples to investigate RNA-level changes

Results:

The analysis revealed a quantifiable, drug-induced change in the overall tRNA charging percentage.

Link between drug exposure and cellular molecular mechanism.
Supporting data-driven decisions in early-stage drug development.