The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei

Xuemin Guo,Jason Carnes,Nancy Lewis Ernst,Kenneth D Stuart,Yue Feng

doi:10.1371/journal.pone.0008913

Abstract

Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by ∼20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C- terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3′-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.

Highlights

The mitochondrial DNA of trypanosomes, called kinetoplast DNA, is composed of tens of maxicircles and thousands of minicircles [1]
Western analysis of the cell samples with KREPA3 monoclonal antibody (MAb) showed that the myc-tagged WT and A3ZFm2 were only detected after the expression of untagged KREPA3 Reg was repressed (R), and their apparent protein levels were lower than that of KREPA3 Reg; neither A3ZFm1 nor A3ZFm1&2 proteins were detected after repression of KREPA3 Reg expression (R) (Fig. 1B)
KREPA3 with mutation of ZF1, which could only be examined in cells exclusively expressing the TAPtagged KREPA3ZFm1 [49], resulted in accumulation of partially edited ATP synthase 6 mRNA in which the editing was restricted to the most 39 region that could be specified by a single guide RNAs (gRNAs) (Fig. 5)

Summary

Introduction

The mitochondrial DNA of trypanosomes, called kinetoplast DNA, is composed of tens of maxicircles and thousands of minicircles [1]. Twelve maxicircle transcripts undergo the post-transcriptional uridine (U) insertion/deletion RNA editing to create the functional open reading frames (ORFs) [2,3]. Some transcripts are extensively edited throughout their length, such as ATPase subunit 6 (A6) [4], cytochrome oxidase subunit III (COIII) [5], and ribosomal protein S12 (RPS12) [6]; while editing is restricted to smaller domains in other transcripts, such as apocytochrome b (CYb) [7,8], cytochrome oxidase subunit II (COII) [9], and maxicircle unidentified reading frame 2 (MURF2) [10]. Little edited CYb and COII mRNAs are present in the mammalian bloodstream slender form (BF) stages of T. brucei, but substantial amounts of these edited RNAs are present in insect procyclic forms (PFs) [7,9]. The 39 domain of NADH dehydrogenase 7 (ND7) transcripts are only partially edited in BFs but fully edited in PFs [11]

Methods

Results

Conclusion