Trypanosomatidae is a family of flagellated proto- zoans. A variety of Trypanosomatidae species from the Leishmania and Trypanosoma genera are responsible for various human diseases that lead to millions of deaths

Trypanosomatidae is a family of flagellated protozoans. A variety of Trypanosomatidae species from the Leishmania and Trypanosoma genera are responsible for various human diseases that lead to millions of deaths in developing countries. These organisms branch early in the phylogenetic tree and exhibit a single, conserved mechanism for gene expression (Simpson et al. 2006). Protein coding genes are polycistronically transcribed by RNA polymerase II from only a small number of promoter regions and individual nuclear messenger RNA (mRNA) is then generated via two coupled reactions: trans-splicing and polyadenylation (Lebowitz et al. 1993, Mayer & Floeter-Winter 2005). Although transsplicing is a processing event that involves two independently transcribed RNA molecules, spliced leader (SL) RNA and pre-mRNA, the acceptor and donor sequences are identical to those in the majority of cis-spliced transcripts (Liang et al. 2003, Mayer & Floeter-Winter 2005). Trans-splicing processing adds a 39-nt mini-exon (ME) (SL) sequence derived from SL RNA to the 5’ end of virtually all mRNAs. The SL RNA donor sites invariably consist of a GU dinucleotide. This processing occurs at transcribed intergenic regions and the acceptor sites are generally composed of an AG dinucleotide preceded by a polypyrimidine tract (Liang et al. 2003, Mayer & Floeter-Winter 2005, Orlando et al. 2007). The polyadenylation of an upstream gene appears to be coupled to the trans-splicing of a second gene that is typically 200-500 nts downstream (Lebowitz et al. 1993, Hug et al. 1994). High-throughput analyses have shown that the locations of both the trans-splicing and the polyadenylation acceptor sites are quite variable in trypanosomatids and a considerable number of alternative sites have been described (Siegel et al. 2011). Until recently, SL addition trans-splicing was exclusively associated with pre-mRNA processing and other RNA molecules were not considered to be involved in this processing mechanism. However, polyadenylation of non-protein coding genes has been detected in both prokaryotes and eukaryotes (Fleischmann et al. 2004). In trypanosomatids, it has been shown that ε ribosomal RNA (rRNA) fragment precursors (the 3’-most portion of the fragmented large rRNA subunit) are polyadenylated in Leishmania (Viannia) braziliensis and Leishmania (Leishmania) donovani (Decuypere et al. 2005). Moreover, in Trypanosoma brucei, a small number of RNA polymerase II-transcribed tRNAsec precursor transcripts have been shown to exhibit SL ME sequences attached to their 5’ ends (Aeby et al. 2010). Finally, we have identified pre-rRNA molecules that are processed by transsplicing in the 5’ external transcribed spacer (5’ETS) region in four trypanosomatid species [T. brucei, Trypanosoma cruzi, Leishmania (Leishmania) amazonensis and Crithidia fasciculate] and by 5’ETS polyadenylation in L. (L.) amazonensis (Mayer et al. 2012). These results show that such processing events are not restricted to pre-mRNAs in these organisms. In this study, we showed that pre-rRNA trans-splicing is not restricted to the 5’ETS region. In addition, we demonstrated that the internal transcribed spacer (ITS) I region of transcripts in L. (L.) amazonensis is also a possible acceptor region for trans-splicing. Financial support: FAPESP, CNPq + Corresponding author: [email protected] Received 20 April 2012 Accepted 28 July 2012 Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis