The microRNAs in an ancient protist repress the variant-specific surface protein expression by targeting the entire coding sequence.

microRNAs (miRNA) have been detected in the deeply branched protist, Giardia lamblia, and shown to repress expression of the family of variant-specific surface proteins (VSPs), only one of which is expressed in Giardia trophozoite at a given time. Three next-generation sequencing libraries of Giardia Argonaute-associated small RNAs were constructed and analyzed. Analysis of the libraries identified a total of 99 new putative miRNAs with a size primarily in the 26 nt range similar to the size previously predicted by the Giardia Dicer crystal structure and identified by our own studies. Bioinformatic analysis identified multiple putative miRNA target sites in the mRNAs of all 73 VSPs. The effect of miRNA target sites within a defined 3-region were tested on two vsp mRNAs. All the miRNAs showed partial repression of the corresponding vsp expression and were additive when the targeting sites were separately located. But the combined repression still falls short of 100%. Two other relatively short vsp mRNAs with 15 and 11 putative miRNA target sites identified throughout their ORFs were tested with their corresponding miRNAs. The results indicate that; (1) near 100% repression of vsp mRNA expression can be achieved through the combined action of multiple miRNAs on target sites located throughout the ORF; (2) the miRNA machinery could be instrumental in repressing the expression of vsp genes in Giardia; (3) this is the first time that all the miRNA target sites in the entire ORF of a mRNA have been tested and shown to be functional. Author SummaryGiardia lamblia is a protozoan parasite causing the diarrheal disease giardiasis. Variant-specific surface proteins (VSP) in Giardia are likely involved in its evasion of host immune response. Their expression is regulated by microRNAs (miRNA). To determine the full complement of miRNAs in Giardia, three cDNA libraries of Giardia Argonaute associated small RNAs were constructed and analyzed to identify a total of 105 miRNAs. Bioinformatic target identification showed that 102 of the 105 miRNAs find their putative target sites in vsp mRNAs. When only the target sites within the 3 region,100 nts upstream of the stop codon, were tested against their corresponding miRNAs, however, only partial repression of VSP expression was observed. When all the miRNA target sites in the open reading frames of vsp mRNAs were examined, however, they all turned out to be functional. A saturation of them with the corresponding miRNAs resulted in a full repression of VSP expression, suggesting that this is the mechanism of miRNA repression of VSP expression in Giardia. The ability of miRNAs to regulate target sites throughout the entire open reading frame also provides the first indication that all the miRNA target sites in an mRNA are functional.