Mitochondrial RNA processing in trypanosomes

Abstract

The mitochondrial genome of trypanosomes is composed of ∼50 maxicircles and thousands of minicircles. Maxi-(∼25 kb) and mini-(∼1 kb)circles are catenated and packed into a dense structure called a kinetoplast. Both types of circular DNA are transcribed by a phage-like RNA polymerase: maxicircles yield multicistronic rRNA and mRNA precursors, while guide RNA (gRNA) precursors are produced from minicircles. To function in mitochondrial translation, pre-mRNAs must undergo a nucleolytic processing and 3' modifications, and often uridine insertion/deletion editing. gRNAs, which represent short (50-60 nt) RNAs directing editing reactions, are produced by 3' nucleolytic processing of a much longer precursor followed by 3' uridylation. Ribosomal RNAs are excised from precursors and their 3' ends are also trimmed and uridylated. All tRNAs are imported from the cytoplasm and some are further modified and edited in the mitochondrial matrix. Historically, the fascinating phenomenon of RNA editing has been extensively studied as an isolated pathway in which nuclear-encoded proteins mediate interactions of maxi- and minicircle transcripts to create open reading frames. However, recent studies unraveled a highly integrated network of mitochondrial genome expression including critical pre- and post-editing 3' mRNA processing, and gRNA and rRNA maturation steps. Here we focus on RNA 3' adenylation and uridylation as processes essential for biogenesis, stability and functioning of mitochondrial RNAs.

Fig. 1

General outline of mitochondrial RNA processing in trypanosomes. Polycistronic transcripts are produced by mitochondrial RNA polymerase from maxicircle and minicircle components of the kinetoplast DNA. ND and NADH dehydrogenase. CO, cytochrome c oxidase. MURF, mitochondrial unidentified frame. GR, G-rich region. A single trans-acting gRNA (ND2, gMurf1-[II]) is transcribed from the maxicircle. Irrespective of genomic location, pre-gRNAs undergo 3′ nucleolytic processing by a cryptic nuclease via a pathway requiring RET1 activity (Aphasizheva and Aphasizhev, 2010). Processed guide RNAs are stabilized by binding to the gRNA binding complex (GRBC, (Weng et al., 2008;Aphasizheva and Aphasizhev, 2010)) and 3′ uridylated by RET1 TUTase (Aphasizhev et al., 2002;Aphasizhev et al., 2003b), but the order of events is unclear. Pre-rRNAs are also trimmed at the 3′ end and uridylated by RET1 (Aphasizheva and Aphasizhev, 2010). Guide RNA hybridization with pre-edited mRNA activates the editing process, but may also be responsible for transient association of RET1 with 3′ adenylation (KPAP1) complex. Editing events in the 3′ region confer a requirement for the short A-tail as cis-element necessary (Etheridge et al., 2008) and sufficient for mRNA stability (Aphasizheva et al., 2011). The completion of editing, typically at the 5′ region, triggers extension of the pre-existing A-tail into A/U-heteropolymer by KPAF1-2-coordinated, KPAP1/RET1-catalyzed reaction. This final processing event (green arrows) constitutes a rate-limiting step in mitochondrial mRNA processing which facilitates mRNA binding to the small ribosomal subunit (Aphasizheva et al., 2011).

Fig. 2

A. Schematic representation of U-deletion (left) and U-insertion (right) enzymatic cascades catalyzed by RECC1 and RECC2, respectively. RECC – RNA editing core complex. U-insertion and U-deletion subcomplexes are depicted in blue and green, respectively. MP: mitochondrial protein; REX: RNA editing exonuclease; REN: RNA editing endonuclease; REL: RNA editing ligase; RET: RNA editing TUTase; gRNA: guide RNA; anchor: 5-15-nt long double-stranded region formed by the 5′-portion of the gRNA and pre-edited mRNA. Initial endonucleolytic mRNA cleavage occurs at the first unpaired nucleotide upstream of the anchor. B. Polarity of mRNA editing. The 3′-5′ (mRNA) polarity of RNA editing is determined by hierarchical gRNA binding in which editing at the 3′ region often creates a binding site for the next gRNA.

Fig. 3

A model for the editing status-dependent ‘switch’ in short A-tail function. After precursor cleavage, pre-edited mRNA is protected against 3′-5′ degradation by virtue of hypothetical protein factor (X) binding to the 3′ region. Hence, the short A-tail is dispensable for pre-edited mRNA stabilization, and KPAP1 knockdown has no effect on mRNA abundance (Etheridge et al., 2008). Progression of editing through the 3′ region displaces factor X; therefore, mRNA stability becomes dependent on the short A-tail and, most likely, protein factors bound to the short tail, e.g., KPAP1. Thus, KPAP1 knockdown abolishes edited mRNAs. Generation of a new sequence by RNA editing leads to binding of a sequence-specific factor Y, mRNA circularization and recruitment of KPAF1 and 2, and RET1 to the 3′ end. A combination of KPAP1, RET1 and KPAFs is sufficient to induce A/U-tail formation in vitro (Aphasizheva et al., 2011).

Fig. 4

A model for generation of gRNAs and gRNA-like molecules involved in RNA editing and maxicircle precursor processing, respectively. The double-stranded substrate for the processing endonuclease may be generated by overlapping transcripts synthesized from opposite strands. Uridylation likely takes place after unwinding of the gRNA-sized duplex. It is also possible that the otherwise processive RET1 adds only 15-20 Us to GRBC-bound gRNAs and gRNA like molecules. We propose that the first editing step (gRNA-directed mRNA cleavage) and multicistronic precursor cleavage are directed by trans-acting short RNA molecules bound to the gRNA biding complex (GRBC). The editing endonucleases have been identified as RNase III-type enzymes (reviewed in (Aphasizhev and Aphasizheva, 2011), suggesting that members of the same family may participate in RNA-directed pre-mRNA cleavage.