Intrinsic and regulated properties of minimally edited trypanosome mRNAs.

Brianna L Tylec,Yijun Sun,Rachel M Simpson,Laura E Kirby,Laurie K Read,Donna J Koslowsky,Runpu Chen

doi:10.1093/nar/gkz012

Brianna L Tylec, Yijun Sun + Show 5 more

Open Access

https://doi.org/10.1093/nar/gkz012

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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.

Highlights

Trypanosoma brucei belongs to an early branching class of eukaryotes termed Kinetoplastea, several members of which are the causative agents of parasitic diseases in humans and livestock [1,2,3]
We show that guide RNAs (gRNAs) utilization in minimally edited mRNAs is more efficient than for pan-edited mRNAs, and we demonstrate multiple mechanisms of junction formation
One striking finding was that the proportions of fully edited CYb and MURF2 mRNAs in wild-type cells were much higher than those previously found for pan-edited mRNAs RPS12 and ND7-5 [8]

Summary

Introduction

Trypanosoma brucei belongs to an early branching class of eukaryotes termed Kinetoplastea, several members of which are the causative agents of parasitic diseases in humans and livestock [1,2,3]. Kinetoplastids are named after their unique mitochondrial DNA consisting of dozens of concatenated ∼22 kb maxicircles and several thousand ∼1 kb minicircles [4]. Maxicircles contain 18 protein coding genes, 12 of which contain only very short open reading frames that lack homology to known mitochondrial proteins and are not believed to be translated into proteins. The mRNAs transcribed from these 12 genes require uridine (U) insertion/deletion editing to generate translatable mRNAs [5]. Of the 12 edited mRNAs, 9 require modification throughout their lengths and are termed pan-edited and 3 mRNAs are edited over a much smaller region and are called minimally edited. This process is accomplished in concert with several multiprotein subcomplexes including the RNA Editing Substrate Binding Complex (RESC; aka, MRB1 complex), the enzymatic RNA Editing Core Complex (RECC; aka, 20S editosome), and other editing accessory factors [6,7]

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