High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing

Abstract

Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steadystate mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5 ′ , in wild-type Trypanosoma brucei. We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5 ′ UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences.

MRB7260 is essential for productive protein–RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing

“…To more precisely define the step of editing that is impacted by MRB7260, we next examined the effects of MRB7260 depletion on RNA editing at the sequence population level. We used our previously described protocol that defines the regions in which the 3 ′ to 5 ′ progression of editing is paused upon depletion of essential editing factors (see Supplemental Methods; Simpson et al 2016Simpson et al , 2017. Using this method, a library of pre-edited, partially edited, and fully edited sequences was obtained from two MRB7260 RNAi-induced cDNA samples and 10 uninduced control samples, two from this study and 8 from a previous study (Simpson et al 2017).…”

“…Using this method, a library of pre-edited, partially edited, and fully edited sequences was obtained from two MRB7260 RNAi-induced cDNA samples and 10 uninduced control samples, two from this study and 8 from a previous study (Simpson et al 2017). This library was aligned using TREAT (Simpson et al 2016(Simpson et al , 2017. TREAT defines any space between two non-T nucleotides in the cDNA as an editing site (ES), and ESs are numbered from 3 ′ to 5 ′ , the direction in which overall editing progresses (Fig.…”

“…2B). An editing stop site is defined by TREAT as the 5 ′ -most ES that matches the fully edited sequence, and thus any ES 3 ′ of an editing stop site matches the canonical fully edited sequence while the region 5 ′ of the editing stop site contains either pre-edited or junction sequence (Simpson et al 2016). Junctions are variable edited sequences that match neither pre-edited nor fully edited sequence, and are present in partially edited mRNAs between the 3 ′ fully edited and 5 ′ pre-edited regions.…”

“…Junctions are variable edited sequences that match neither pre-edited nor fully edited sequence, and are present in partially edited mRNAs between the 3 ′ fully edited and 5 ′ pre-edited regions. Junctions are present in >95% of RPS12 mRNAs and are hypothesized to be regions of active editing that undergo repeated remodeling (Koslowsky et al 1991;Sturm et al 1992;Ammerman et al 2010;Simpson et al 2016). TREAT designates junctions as extending from the 3 ′ most ES that does not match the fully edited sequence to the 5 ′ most editing site beyond the editing stop site that shows any editing modification.…”

ARSDA: A new approach for storing, transmitting and analyzing high-throughput sequencing data

“…First, many reads do not map exactly to the genomic sequence because of either somatic mutations or sequencing errors, so representing a read by the starting and ending numbers leads to loss of information. Second, RNAediting and processing can be so extensive that it becomes impossible to map a transcriptomic read to the genome (ABRAHAM et al 1988;LAMOND 1988;ALATORTSEV et al 2008;LI et al 2009;SIMPSON et al 2016). Furthermore, there are still many scientifically interesting species that do not have a good genomic sequence available.…”