RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3′ to 5′ on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex.
Researchers at Lomonosov Moscow State University present their analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, their new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. The researchers also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.
Overview of T. cruzi RPS12 transcript editing and reconstruction of the canonical edited product, a well-supported 5′ truncated alternative ORF, and a full-length alternative ORF with an N-terminal shifted reading frame
The number of Ts in the genomic sequence is shown with stacked red bars at top. (A) Visualization of editing states found in at least two reads (light-grey dots). Proportion of reads supporting an editing state is calculated for each site separately and color-coded, with the blue dot being the most supported and black, the next most supported. (B) Absolute coverage bar graph with proportions of U-indel insertion edited (blue), deletion edited (pink), or never-edited reads (green). The proportion of edited reads at each site is also shown with black vertical lines. Y-axis values for both metrics are provided. (C) ORFs visualized as paths through editing states. Translatable editing states (those included into at least one ORF >60 aa in length) are boxed in green and overlaid with the canonical ORF (red) and alternative ORFs (black and blue). Where editing differs in the black and blue ORF compared with the canonical ORF, it is indicated by dots between panels C and D. Red triangles, start codons; red squares, stop codons. (D) Cloud coverage diagrams of editing states at each edited site. Each read is plotted as a semi-transparent dot. The canonical and alternative ORFs shown in C are also plotted. Blue horizontal lines in B, C and D indicate a domain of editing that remains unedited in the ‘blue’ truncated alternative product.
Availability – T-Aligner’s code is available at GitHub [https://github.com/jalgard/T-Aligner3].