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The parasite Trypanosoma brucei is the causative agent of sleeping sickness and cycles between insect and mammalian hosts. The parasite appears to lack conventional transcriptional regulation of protein coding genes, and mRNAs are processed from polycistronic transcripts by the concerted action of trans- splicing and polyadenylation. Regulation of mRNA function is mediated mainly by RNA binding proteins affecting mRNA stability and translation. In this study, we describe the identification of 62 non-coding (nc) RNAs that are developmentally regulated and/or respond to stress. We characterized two novel anti-sense RNA regulators (TBsRNA-33 and 37) that originate from the rRNA loci, associate with ribosomes and polyribosomes, and interact in vivo with distinct mRNA species to regulate translation. Thus, this study suggests for the first-time anti-sense RNA regulators as an additional layer for controlling gene expression in these parasites., Competing Interests: The authors declare no competing financial interests., (© 2020 The Authors.)