Expression studies on clustered trypanosomatid box C/D small nucleolar RNAs

We analyzed three chromosomal loci of the trypanosomatid Leptomonas colloso ma encoding box C/D small nucleolar RNAs (snoRNAs). All the snoRNAs that we re analyzed here carry two sequences complementary to rRNA target sites and obey the +5 rule for guide methylation. Studies on transgenic parasites ca rrying the snoRNA-2 gene in the episomal expression vector (pX-neo) indicat ed that no promoter activity was found immediately adjacent to this gene. D eleting the flanking sequences of snoRNA-2 affected the expression; in the absence of the 3'-flanking (but not 5'-flanking) sequence, the expression w as almost completely abolished. The snoRNA genes are transcribed as polycis tronic RNA. All snoRNAs can be folded into a common stem-loop structure, wh ich may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster, was expressed when cloned into the episomal v ector, suggesting that each gene within a cluster is individually processed . Studies with permeable cells indicated that snoRNA gene transcription was relatively sensitive to alpha -amanitin, thus supporting transcription by RNA polymerase II. We propose that snoRNA gene expression, similar to prote in-coding genes in this family, is regulated at the processing level.