The Identification Of Pseudogene-derived SiRNAs, NATs And The Function Of MRB1 Complex Subunits In Trypanosoma Brucei

Trypanosoma brucei, the pathogen of sleeping sickness (African trypanosomiasis) in human and of Nagana in animals/livestock causing serious public health problem and significant economic loss in the endemic regions particularly sub-Sahara areas of African. As an early evolutionarily ancient protozoan, T. brucei has become a good model for the study of the origin of eukaryotics. T. brucei undergoes a complex life cycle within the mammalial host and insect vector. However, their genes are transcribed constitutively in different stages and are regulated post-transcriptionally. So far as we know, unfortunately, the regulatory mechanism and the molecules involved in these transcriptions are largely unknown.Pseudogenes are considered to be dysfunctional. However, more and more evidence indicated that they may have important functions in gene regulation. Here, we found a cluster of siRNAs derived from pseudogenes of T. brucei brucei using high through-put analysis and demonstrated that these pseudogene-derived siRNAs suppress gene expression through RNA interference.Natural antisense transcripts (NATs) have been shown to play important roles at the level of post-transcription and generated siRNAs to regulate gene expression. So far as we know, there is no report regaring the identification of NATs in trypanosomatids by analysis of genome. In this study, the analysis of NATs in genome-wide and nat-siRNAs based on transcripts annotation as well as the deep-sequencing of T. brucei brucei were carried out. More NAT pairs with noncoding transcripts were found in the bloodstream form than in the procyclic from. The results from the analysis indicated that the NATs are widespread distributed on the whole genome of T. brucei brucei but absent from the mitochondrial (i.e. kDNA) genome. The fact of small RNAs originated from the overlapping regions of NATs revealed that they have highly similar characteristics as T. brucei siRNAs and considered that they regulate the expression of genes through RNAi pathway according to the experimental evidence. Results from the Gene Ontology analysis further revealed that these nat-siRNAs can contribute to the differentiation of T. brucei brucei indeed.The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in T. brucei, which have been proved to function on RNA editing. Results from the RNAi silencing of the two newly characterized protein subunits of MRB1, guide RNA associated proteins (GAPs) 1 and 2, show that they are essential for cell growth in the bloodstream forms of this parasite. Furthermore, down-regulation of their gene expression can result in inhibition of the synthesis of the maxicircle-encoded acting gRNA. Moreover, Western Blot results showed that GAP1 and 2 are concentrated into the mitochondrion fraction.Taken together, the discovery of pseudogene-derived siRNAs with regulatory roles and the identification of NATs and nat-siRNAs not only highlight the novel function of pseudogenes, but also enhance the derivation of siRNAs in African trypanosome and provide a novel direction to study the origin of small RNAs in eukaryota. In addition, the difference of NATs between different stages and the potential RNA editing in the bloodstream forms will offer novel interpretation to the differentiation of T. brucei as well.