Giardia lamblia, an ancient flagellated protist, is an infectious pathogen that causes diarrheal diseases throughout the world and the most common cause of water-borne outbreaks of diarrhea in the United States. Among the many unique biological features of this pathogen, the unusually short 5'-untranslated region (UTR) of 0 to 14 nucleotides in the mRNAs is perhaps the most intriguing, because a 5'-UTR of less than 20 nucleotides is known to cause leaky ribosomal scanning resulting in erroneous and inefficient protein synthesis in other eukaryotes. In our analysis of an internal ribosomal entry site (IRES) in the Giardia-virus transcript, we identified the AUG start codon at the center of an unstructured 31 nucleotide stretch between two stem-loops, suggesting a direct recruitment of small ribosomal subunit onto the AUG codon without ribosomal scanning. The absence of initiation factors eIF4G, eIF4B and eIF4H from Giardia further supports such a postulation. Expression of capped reporter transcripts in Giardia indicates that a 5'-UTR of 1 to 9 nucleotides results in optimal translation, whereas a 5'-UTR going beyond 14 nucleotides decreases the translation efficiency with its increasing length. When multiple AUGs are present, it is invariably the first one closest to the cap that initiates translation. It is thus clear that ribosomal scanning is absent from Giardia and a simple cap-N-AUG is apparently adequate for starting the formation of a translation initiation complex. In the next granting period, we plan to thoroughly characterize this unique complex with the anticipation that its distinctive properties will make it an attractive target for anti-giardiasis chemotherapy. We shall also look into the likely simple complex that initiates translation from the viral IRES and use it as a simple model for an in-depth understanding of the mechanism of translation initiation in a eukaryote. Furthermore, Giardia is known to contain a substantial amount of small antisense RNAs, but the RNA interference machinery is apparently missing from this organism. We plan to explore the possible presence of a microRNA (miRNA)-mediated translation repression in Giardia by first identifying the potential miRNAs and their probable targets and then testing the possible miRNA inhibition of translation of the target mRNA. It is anticipated that fulfillment of these three aims listed in this application will immensely advance our understanding of the regulation of translation in this ancient organism and provide opportunities for therapeutic attack on the disease it causes. PROJECT NARRATIVE: Giardia lamblia is an infectious pathogen that causes diarrheal diseases throughout the world and has been classified by the U.S. Government as one of the potential agents for bioterrorism. Our research on this pathogen has indicated that it may have a very simple machinery for protein synthesis that does not require an un-translated region at the beginning end of messenger RNA to allow ribosomes to scan for the appropriate initiating point for protein synthesis. This remarkably simple mechanism of initiating protein synthesis could provide a good opportunity for selective killing of Giardia without affecting the human host. We are thus planning on identifying this initiating protein complex for protein synthesis in Giardia and eventually targeting it for selective inhibition. We have also accumulated enough evidence to suggest that some of the peculiar small antisense RNAs in Giardia may play the crucial function of microRNA in regulating protein synthesis. We intend to verify this interesting possibility and further develop it into another opportunity for anti-giardiasis chemotherapy.
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