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Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5' capped mini-exon which is linked to the 3' cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 x 10(5) molecules/cell) and depletion to approximately 10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (approximately 1-2 x 10(4) molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.

Background Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA molecules. In this study, we performed a systematic genome-wide in silico screen to search for novel small ncRNAs in the genome of Trypanosoma brucei using techniques of comparative genomics. Results In this study, we identified by comparative genomics, and validated by experimental analysis several novel ncRNAs that are conserved across multiple trypanosomatid genomes. When tested on known ncRNAs, our procedure was capable of finding almost half of the known repertoire through homology over six genomes, and about two-thirds of the known sequences were found in at least four genomes. After filtering, 72 conserved unannotated sequences in at least four genomes were found, 29 of which, ranging in size from 30 to 392 nts, were conserved in all six genomes. Fifty of the 72 candidates in the final set were chosen for experimental validation. Eighteen of the 50 (36%) were shown to be expressed, and for 11 of them a distinct expression product was detected, suggesting that they are short ncRNAs. Using functional experimental assays, five of the candidates were shown to be novel H/ACA and C/D snoRNAs; these included three sequences that appear as singletons in the genome, unlike previously identified snoRNA molecules that are found in clusters. The other candidates appear to be novel ncRNA molecules, and their function is, as yet, unknown. Conclusions Using comparative genomic techniques, we predicted 72 sequences as ncRNA candidates in T. brucei. The expression of 50 candidates was tested in laboratory experiments. This resulted in the discovery of 11 novel short ncRNAs in procyclic stage T. brucei, which have homologues in the other trypansomatids. A few of these molecules are snoRNAs, but most of them are novel ncRNA molecules. Based on this study, our analysis suggests that the total number of ncRNAs in trypanosomatids is in the range of several hundred.

In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F – eIF4E, eIF4A and eIF4G – to the monomethylated cap present on the 5′ end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5′ end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae . However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5′ ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es ( Lm EIF4E1-4), two eIF4As ( Lm EIF4A1-2) and five eIF4Gs ( Lm EIF4G1-5). We report the initial characterisation of Lm EIF4E1-3, Lm EIF4A1-2 and Lm EIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. Lm EIF4A1 and Lm EIF4E3 are very abundant, Lm EIF4G3 is moderately abundant and Lm EIF4E1/ Lm EIF4E2/ Lm EIF4A2 are rare or not detected. In cap-binding assays, only Lm EIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that Lm EIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A ( Lm EIF4A1/ Lm EIF4A2) and eIF4G ( Lm EIF4G1-3) homologues. Only Lm EIF4G3, via the HEAT domain, bound specifically both to Lm EIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.