Intrinsic and regulated properties of minimally edited trypanosome mRNAs

Abstract

Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing. We show that CYb and MURF2 mRNAs exhibit barriers to editing progression that differ from those previously identified for pan-edited mRNAs, primarily at initial gRNA usage and gRNA exchange. We demonstrate that mis-edited junctions arise through multiple pathways including mis-alignment of cognate gRNA, incorrect and sometimes promiscuous gRNA utilization and inefficient gRNA anchoring. We then examined the roles of accessory factors RBP16 and MRP1/2 in maintaining edited CYb and MURF2 populations. RBP16 is essential for initiation of CYb and MURF2 editing, as well as MURF2 editing progression. In contrast, MRP1/2 stabilizes both edited mRNA populations, while further promoting progression of MURF2 mRNA editing. We also analyzed the effects of RNA Editing Substrate Binding Complex components, TbRGG2 and GAP1, and show that both proteins modestly impact progression of editing on minimally edited mRNAs, suggesting a novel function for GAP1.

MRB10130 is a RESC assembly factor that promotes kinetoplastid RNA editing initiation and progression

“…that arise upon MRB7260 RNAi and again observed a statistically significant overlap with EPSs in MRB10130 depleted cells (P = 0.002) ( Fig. 2C; Tylec et al 2019). EPSs in CYb mRNA upon MRB10130 and TbRGG2 knockdown did not overlap (P = 0.50) (Simpson et al 2017).…”

“…We can then further analyze editing progression by determining the editing stop site in each mRNA, which is defined as the final (5 ′ most) ES that matches the canonical fully edited sequence correctly (Table 1; Simpson et al 2016). At a population level, TREAT allows us to define exacerbated pause sites (EPSs; Table 1); EPSs are editing stop sites at which canonical editing pauses to a significant extent in a given knockdown cell line compared to uninduced controls (P < 0.05, q < 0.05) (Simpson et al 2017;McAdams et al 2018;Tylec et al 2019). Additionally, we can analyze the lengths and sequences of misedited junctions 5 ′ of EPSs to provide insights into specific editing defects that arise when distinct editing factors are depleted (Table 1; Simpson et al 2017;McAdams et al 2018;Tylec et al 2019).…”

Parallel monitoring of mRNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

“…This is consistent with earlier studies on A6 32 and with studies on two other pan-edited transcripts, ND7 and RPS12 32,33 . Higher fractions (up to 30%) of fully decreases with the number of editing events 33,54 . COXIII and A6 have more editing events than the mRNAs sequenced so far providing an explanation for the large fraction of unedited sequences.…”

“…edited RNAs are only found in minimally edited mRNAs54 . With our experimental set-up, it is impossible to distinguish RNA editing intermediates from fully edited or fully unedited RNA species.…”

Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

“…RNA from PCF 29–13 and BSF 427, 90–13, SM-1 and SM-2 cell lines was isolated, DNase-treated, and converted to gene-specific cDNA using primers specific for the editing domain of the minimally-edited cytochrome b (CYb), the 3′ region of the editing domain for cytochrome oxidase subunit III (COIII), and the 3′ region of the editing domain for ATPase subunit 6 (A6) mRNAs. The primer sequences used for the generation of these cDNAs are listed in the supplemental data ( Supplementary Table S1 ) ( 74 ). The cDNAs were PCR-amplified within the linear range to ensure that subpopulations of short sequences were not disproportionately amplified in our samples ( 42 ).…”