Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica

Abstract

In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh_U3snoRNA boxes A and A′ with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5′-hinge and 3′-hinge sequences of Eh_U3 snoRNA with the 5′-ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human.