Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes

Abstract

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Keywords: RNA decay; RNA editing; Trypanosoma; kinetoplast; mitochondria; polyadenylation.

Figure 1.. Schematic Diagram of Mitochondrial RNA Processing in Trypanosoma brucei.

The flow of processing reactions does not imply an experimentally established timing of these events. For example, the rRNA assembly into the ribosome or 5′ pyrophosphate removal from mRNA may occur cotranscriptionally. Likewise, the mRNA may be edited prior to completion of 3′–5′ trimming and 3′ adenylation. Abbreviations: MPsome, mitochondrial 3′ processome; PPi, inorganic pyrophosphate; PPsome, 5′ pyrophosphate processome; RECC, RNA-editing catalytic complex; RESC, RNA-editing substrate-binding complex.

Figure 2.. Guide RNA-Processing Model.

Bidirectional transcription of a guide (g)RNA gene from inverted repeats (IRs) generates overlapping sense and antisense precursors. In Trypanosoma brucei, a minicircle typically contains up to five gRNA genes. The mitochondrial 3′ processome (MPsome) catalyzes three sequential processing reactions: primary precursor uridylation, processive precursor degradation, and secondary uridylation of the mature gRNA. Primary uridylation by KRET1 stimulates hydrolytic activity of KDSS1. The MPsome stochastically pauses at 10–12 nt from sufficiently stable duplex regions, which provides a kinetic window for secondary uridylation. This step may disengage the MPsome from the duplex intermediate. Double-stranded RNA likely undergoes active unwinding before mature gRNA can be sequestered by the RESC complex and delivered into the editing pathway. Abbreviation: RESC, RNA-editing substrate-binding complex.

Figure 3.. A Model for mRNA Quality Control by Pentatricopeptide Repeat (PPR) RNA-Binding Proteins.

We propose that mRNA stability, terminal modifications, and translational activation is largely determined by sequence-specific PPR RNA-binding proteins. Displacement of polyadenylation factor KPAF3 and the 5′ pyrophosphate processome (PPsome) subunit MERS2 by initiating and final editing events, respectively, appears to monitor editing progression and enables temporally separated modifications of the termini. The mRNA circularization is postulated to occur upon KPAF4 binding to a nascent A-tail and ensuing interaction with the PPsome. Displacement of the latter from the 5′ region by final editing events may provide access to KPAF1/2, which stimulates postediting A/U-tailing of fully edited mRNAs, leading to their translational activation. Abbreviation: MPsome, mitochondrial 3′ processome; PPi, inorganic pyrophosphate.

Figure 4.. Schematic Diagram of RNA-Editing Catalytic Complex (RECC) Isoforms and Editing Reactions.

Trans-guided insertion and deletion, and cis-guided insertion substrates are juxtaposed with corresponding endonuclease modules and 12 common core proteins, with catalytic pathways outlined. Abbreviations: 5′ anchor, 5′ part of the guideRNA that hybridizes with pre-edited mRNA; A, KREPA; B, KREPB; SC, subcomplex. See Table 1 for protein designations.