Application of SHAPE reveals in vivo RNA folding under normal and growth-stressed conditions in the human parasite Entamoeba histolytica.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a versatile sequence independent method to probe RNA structure in vivo and in vitro. It has so far been tried mainly with model organisms. We show that cells of Entamoeba histolytica, a protozoan parasite of humans are hyper-sensitive to the in vivo SHAPE reagent, NAI, and show rapid loss of viability and RNA integrity. We optimized treatment conditions with 5.8S rRNA and Eh_U3 snoRNA to obtain NAI-modification while retaining RNA integrity. The modification patterns were highly reproducible. The in vivo folding was different from in vitro and correlated well with known interactions of 5.8S rRNA with proteins in vivo. The Eh_U3 snoRNA also showed many differences in its in vivo versus in vitro folding, which correlated with conserved interactions of this RNA with 18S rRNA and 5'-ETS. Further, Eh_U3 snoRNA obtained from serum-starved cells showed an open 3'-hinge structure, indicating disruption of 5'-ETS interaction. This could contribute to the observed slow processing of pre-rRNA in starved cells. Our work shows the applicability of SHAPE to study in vivo RNA folding in a parasite and will encourage the use of this reagent for RNA structure analysis in other such organisms.