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

Abstract

Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

The Complete Mitochondrial DNA of Trypanosoma cruzi: Maxicircles and Minicircles

“…Mitochondrial metabolism and gene expression are highly regulated to deal with all those complicated environmental changes across the complete life cycle alternating between the mammalian host and insect vector, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation, as has been described in other closely related Trypanosomatids ( Smith et al., 2020 ). To our knowledge, it is the first time describing a large repertoire of complete T. cruzi minicircle sequences.…”

“…Minicircles are exclusive to Trypanosomatids and they are directly involved in U-insertion/deletion editing system as they encode guide RNAs (gRNAs) ( Simpson et al., 2003 ; Aphasizhev and Aphasizheva, 2014 ; Smith et al., 2020 ). Moreover, it is suggested that both molecule populations are heterogeneous in the cell, showing strain-specific variations ( Westenberger et al., 2006 ; Messenger et al., 2012 ).…”

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing

“…The region 5’ of an editing stop site can either be pre-edited or a junction. Junctions are areas of mis-editing, in which the sequences match neither the pre-edited nor fully edited sequence, and they are found in a majority of our partially edited A6, COIII and RPS12 mRNAs ( Table 1 ) (Simpson et al 2016; Smith et al 2020). TREAT defines junctions as extending from the 3’ most ES that does not match the fully edited sequence to the 5’ most editing site that shows any editing modification.…”

“…TREAT v0.03 (Simpson et al 2017) was used in this study. All reads were aligned to the published pre-edited and fully edited A6 and COIII mRNA sequences (Smith et al 2020), with the modification of COIII mRNA as described above. The number of standard reads (sequences with no non-T mismatches) and non-standard reads (sequences with non-T mismatches) are listed in ( Table S1 ).…”

“…Libraries were obtained from two replicates each of induced RESC13 and RESC14 RNAi samples, and compared to two uninduced RESC14 or RESC13 RNAi cells, respectively, as well as five PF 29-13 cells from another study (compared to seven uninduced samples total) (Smith et al 2020). Sequences were normalized to 100,000 reads and aligned using the TREAT Sortino 8 algorithm, as previously described (Simpson et al 2016;Simpson et al 2017).…”