Trypanosomatid mitochondrial RNA editing: dramatically complex transcript repertoires revealed with a dedicated mapping tool

Abstract

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. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our 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. We 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.

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

“…Gene expression levels of mitochondrial RNAs were first compared between replicating amastigotes (collected at the same time that infection metrics were determined) and the T. cruzi culture originally used to infect these cells. As only 20 total protein coding and rRNA loci exist in the mitochondrion, and deep sequencing read populations of T. cruzi mitochondrial genomes contain relatively few that are fully canonically edited (Gerasimov et al, ), reverse transcription—quantitative polymerase chain reaction (RT‐qPCR) is the best method to compare trypanosome mitochondrial RNA abundances for this study. We first examined the abundances of eight RNAs not affected by uridine insertion/deletion editing.…”

Common Structural Patterns in the Maxicircle Divergent Region of Trypanosomatidae

“…Some minor length variations between the CRs can be explained by the editing patterns (variable length of the edited domains) of some cryptogenes. As such, the shortest CR sequences were documented in Trypanosoma spp., where cryptogenes undergo the most extensive RNA editing [4,5], while the longest CR sequences were in monoxenous trypanosomatids, whose cryptogenes have reduced editing domains [37][38][39]. Table 1.…”

Gene expression to mitochondrial metabolism: Variability among cultured Trypanosoma cruzi strains

“…For a wider perspective, we took advantage of high-throughput RNA sequencing read samples from CL Brener, Esmeraldo, and Sylvio X10 exponentially replicating epimastigotes that we have collected for mitochondrial transcriptome analysis. Because this purpose requires specialized sample preparation [ 38 ], reads may contain biases typically not found in high-throughput sequencing read populations. Nevertheless, samples (all harvested during exponential growth) from all strains were identically processed and can be used to identify strain variation in gene expression of classes of metabolic genes.…”

“…Reads from libraries used for differential expression analysis are the total RNA-derived Sylvio X10 reads described in Gerasimov et al [ 38 ], and Esmeraldo and CL Brener strain reads obtained identically at the same time (part of the same dataset). Reads were trimmed for quality and of adaptor sequences using Trimmomatic version 0.36 [ 39 ].…”