A Novel Class of Developmentally Regulated Noncoding RNAs in Leishmania

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Abstract
Leishmania is a protozoan parasite that causes serious morbidity and mortality in humans worldwide. The ability of these parasites to survive within the phagolysosomes of mammalian macrophages is dependent on the developmental regulation of a variety of genes. Identifying genomic sequences that are preferentially expressed during the parasite's intracellular growth would provide new insights about the mechanisms controlling stage-specific gene regulation for intracellular development of the parasite. Using a genomic library that differentially hybridized to probes made from total RNA from Leishmania infantum amastigote or promastigote life cycle stages, we identified a new class of noncoding RNAs (ncRNAs) ranging from approximately 300 to 600 nucleotides in size that are expressed specifically in the intracellular amastigote stage. These ncRNAs are transcribed by RNA polymerase II from genomic clusters of tandem head-to-tail repeats, which are mainly located within subtelomeric regions. Remarkably, both the sense and antisense orientations of these ncRNAs are transcribed and are processed by trans splicing and polyadenylation. The levels of antisense transcripts are at least 10-fold lower than those of the sense transcripts and are tightly regulated. The sense and antisense ncRNAs are cytosolic as shown by fluorescence in situ hybridization studies and cosediment with a small ribonucleoprotein complex. Amastigote-specific regulation of these ncRNAs possibly occurs at the level of RNA stability. Interestingly, overexpression of these ncRNAs in promastigotes, as part of an episomal expression vector, failed to produce any transcript, which further highlights the instability of these RNAs in the promastigote stage. This is the first report describing developmentally regulated ncRNAs in protozoan parasites.