Disruption of a Gene Encoding a Novel Mitochondrial DEAD-Box Protein in Trypanosoma brucei Affects Edited mRNAs

Abstract

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.RNA editing of mitochondrial pre-mRNAs in kinetoplastid protozoa is a unique form of posttranscriptional gene transcript maturation. The process is characterized by the sitespecific insertion and deletion of exclusively uridylate residues, thereby creating functional mRNA molecules for translation (reviewed in reference 19). RNA editing relies on small, metabolically stable RNA molecules known as guide RNAs (gRNAs), which act as templates in the process (6,44,45). gRNAs probably initiate the processing reaction by the formation of a short intermolecular duplex structure with the pre-mRNA, located proximal to the sequence domain to be edited. Bordering this helical segment, unpaired gRNA nucleotides then specify via base pairing the U insertion event with free UTP as the substrate (6, 29). Non-base-paired uridylates in the pre-mRNA are deleted. The reaction seems to occur within a high-molecular-mass complex (9, 39), involving a cascade of enzymatic activities (15) such as gRNA-directed endonuclease, terminal uridylyl transferase, terminal uridylyl nuclease, and RNA ligase (12; reviewed in reference 19). Although both pre-mRNAs and gRNAs have been identified in stable complexes with mitochondrial proteins (4; reviewed in references 18 and 30), no polypeptide has been shown to date to directly participate in the editing process.By analogy to other biochemical processes that are catalyzed by macromolecular ribonucleoprotein complexes, it is likely that RNA editing is a highly dynamic process. The participating RNA and protein molecules probably have to undergo a defined series of conformational changes not only during the assembly of the functional particle but also as the reacti...

Proteomics and electron microscopic characterization of the unusual mitochondrial ribosome-related 45S complex in Leishmania tarentolae

“…An additional PPR protein (LmjF21.1620) was identified by a single hit in the dimeric 45S complexes (data not shown) but its relationship with the complex is unclear. The detected helicase is different from the mHel61 DEAD-box enzyme potentially implicated in RNA editing [46]. It is possible that the ribosomal PPR proteins and the helicase mediate interactions between mRNAs and translation machinery, but this hypothesis still needs to be investigated.…”

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

“…In fact, the evidence that the largest effect of REH1 down-regulation on editing is on the pan-edited A6 and CR3 mRNAs suggests that the in vivo activity of REH1 is substrate-specific and perhaps is regulated by transient binding of cofactors. Along this line, no detectable in vitro unwinding activity had been observed with T. brucei gradient fractions that showed a peak of Hel61 (REH1) by blot analysis (21) nor in the Lm REH1 SAP pull-down (Fig. S7B).…”

“…However, gene disruption had no effect on either an experimentally observed mito- chondrial RNA unwinding activity (20) or on full cycle in vitro editing reactions (21). Furthermore, the observed RNA unwinding activity did not cosediment with Hel61 (21). The functional role of Hel61 and the mechanism of gRNA displacement were not clear.…”

“…An RNA helicase has been suggested to be involved in this process by displacing the initial gRNA, thereby allowing the adjacent upstream gRNA to form an anchor duplex with the edited sequence (19). Two trypanosome mitochondrial DExD/H-box proteins have been identified (14,16,20,21) and shown to be involved in RNA editing. RNA editing helicase 2 or REH2 is a component of GRBC or MRB1 complexes and is involved in gRNA biogenesis (13,14,16).…”

“…Hel61, another DEAD-box protein, was shown to be involved in RNA editing by a gene disruption experiment in T. brucei, which produced a slow growth phenotype and affected editing efficiency, and ectopic reexpression of Hel61 rescued a partially restored editing phenotype (21). However, gene disruption had no effect on either an experimentally observed mito- chondrial RNA unwinding activity (20) or on full cycle in vitro editing reactions (21). Furthermore, the observed RNA unwinding activity did not cosediment with Hel61 (21).…”