A comprehensive collection of annotations to interpret sequence variation in human mitochondrial transfer RNAs

Abstract

Background: The abundance of biological data characterizing the genomics era is contributing to a comprehensive understanding of human mitochondrial genetics. Nevertheless, many aspects are still unclear, specifically about the variability of the 22 human mitochondrial transfer RNA (tRNA) genes and their involvement in diseases. The complex enrichment and isolation of tRNAs in vitro leads to an incomplete knowledge of their post-transcriptional modifications and three-dimensional folding, essential for correct tRNA functioning. An accurate annotation of mitochondrial tRNA variants would be definitely useful and appreciated by mitochondrial researchers and clinicians since the most of bioinformatics tools for variant annotation and prioritization available so far cannot shed light on the functional role of tRNA variations.

Results: To this aim, we updated our MToolBox pipeline for mitochondrial DNA analysis of high throughput and Sanger sequencing data by integrating tRNA variant annotations in order to identify and characterize relevant variants not only in protein coding regions, but also in tRNA genes. The annotation step in the pipeline now provides detailed information for variants mapping onto the 22 mitochondrial tRNAs. For each mt-tRNA position along the entire genome, the relative tRNA numbering, tRNA type, cloverleaf secondary domains (loops and stems), mature nucleotide and interactions in the three-dimensional folding were reported. Moreover, pathogenicity predictions for tRNA and rRNA variants were retrieved from the literature and integrated within the annotations provided by MToolBox, both in the stand-alone version and web-based tool at the Mitochondrial Disease Sequence Data Resource (MSeqDR) website. All the information available in the annotation step of MToolBox were exploited to generate custom tracks which can be displayed in the GBrowse instance at MSeqDR website.

Conclusions: To the best of our knowledge, specific data regarding mitochondrial variants in tRNA genes were introduced for the first time in a tool for mitochondrial genome analysis, supporting the interpretation of genetic variants in specific genomic contexts.

Keywords: Annotation and prioritization tools; Bioinformatics analysis; Mitochondrial genomics; NGS; tRNA sequence variation.

Fig. 1

Schematic representation of the four types of human mitochondrial tRNAs. The four types of human mt-tRNAs are shown. Green circles represent all the nucleotide positions involved in post-transcriptional modifications in each tRNA. Blue circles indicate nucleotide positions involved in tertiary folding with interactions represented by lines. Red circles represent nucleotide positions involved in tertiary folding and subject to post-transcriptional modifications. All the stems (A-stem, T-stem, C-stem, D-stem) and loops (T-loop, V-loop, C-loop, D-loop) of cloverleaf secondary regions are also shown

Fig. 2

Overview of the usage of mitochondrial tracks at MSeqDR GBrowse. MSeqDR website provides access to a GBrowse useful to visualize genomics data. Users can upload the four tracks generated in this work in the “Custom Tracks” section of the browser (a). For the sake of simplicity, the only “MT-patho.RNA” track is here shown, including data about pathogenic variants in mt-tRNA and mt-rRNA genes. The custom track can be selected, totally or partially (only transitions, transversions, insertions or deletions, b) and then visualized in the browser (c) where users can search for a specific genomic region of interest. Eventually, detailed information can be shown by clicking on a specific variant site (d)