DEAD-box protein HEL64 from Trypanosoma brucei: subcellular localization and gene knockout analysis.

RNA helicases are molecules that play a central role in the control of ribonucleic acid metabolism. Only two putative RNA helicase genes of the DEAD-box family have been identified in the protozoan parasite Trypanosoma brucei brucei. One of these genes is HEL64, a single-copy gene of unknown function. In this study we conducted targeted gene-disruption experiments of the HEL64 locus with the aim of identifying a phenotype that would suggest a function for the encoded protein. It is likely that HEL64 is an essential gene in insect-stage trypanosomes, since all attempts to create a HEL64 double-allele knockout cell line failed. Instead, we obtained a mutant derived from an unusual recombination event that nonetheless contained a functional copy of the gene. One allele of HEL64 is sufficient for the survival of the parasite. Single-allele knockout mutants showed no gross change in cell morphology and multiplied with a cell-doubling time identical to that of wild-type trypanosomes. Though HEL64 has high sequence homology with the nuclear DEAD-box protein p68, it is localized in the cytosol of trypanosomes and, thus, cannot be a homologue of p68 as previously suggested. Since the protein did not cross-hybridize with an anti-eIF-4A antibody, we excluded the possibility that HEL64 might be a homologue of the translation initiation factor eIF-4A. Although the function of HEL64 remains unknown, the present data indicate that the encoded DEAD-box protein plays an important role during the insect life-cycle stage of the parasite.

Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

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.

Different Trypanosoma brucei guide RNA molecules associate with an identical complement of mitochondrial proteins in vitro.

RNA editing is a mitochondrial transcript maturation process which evolved in kinetoplastid protozoa. It entails the insertion and deletion of exclusively uridine nucleotides directed by gRNAs into pre-mRNAs. Other participating components are not currently known. The aim of this study was to identify mitochondrial proteins that are in direct physical contact with gRNAs thereby possibly involved in the editing reaction. At low monovalent cation concentration (30 mM KCl) 8 polypeptides with apparent molecular weights ranging from 124 to 9 kDa specifically cross-linked to gRNAs. Three of the proteins, 90, 21, and 9 kDa in size, were able to bind at higher salt concentrations (> or = 100 mM) indicating an enhanced affinity to the gRNA molecules. No cross-links were identified at > or = 250 mM KCl. Four gRNAs, specific for different editing domains of the ATPase 6 and ND7 pre-mRNAs, were in contact with the same set of mitochondrial polypeptides suggesting the assembly of an identical RNP complex that does not include pre-mRNA molecules. The binding of the 90 kDa protein was sensitive to the presence of U-nucleotides at the 3'-end of the gRNAs and could specifically be blocked by modifying free sulfhydryl groups. The interaction with the 124 kDa polypeptide was inhibited by vanadyl ribonucleosides, implicating a role for 2', 3' hydroxyl groups in the gRNA-protein interaction.