Estructura bàsica d’un KPI

The field of ribonucleic acid (RNA) biology has become an exciting research area within the last twenty years as an increasing number of non-coding functions have been unveiled for these molecules. The functional repertoire of the ‘small RNA’ universe known in the early nineties -with roles in translation (tRNA and 5S RNA), pre-mRNA processing (by small nuclear RNAs), and protein translocation across the endoplasmatic reticulum (7SL RNA)- experienced a dramatic

expansion after the discovery of 20-30 nucleotide-long small silencing RNA molecules (ssRNA) able to switch off gene expression: the so-called pathway of RNA interference (RNAi). It was characterised for the first time in plants (Napoli, 1990; van der Krol et al., 1990), termed RNAi and assigned to double stranded RNA in the nematode Caenorhabditis elegans (Fire et al., 1998), and was soon described for many other eukaryotes, including Trypanosoma brucei (Ngô et al., 1998). Two main types of ssRNAs were identified in protozoa: small interfering RNA (siRNA), binding specific mRNAs and targeting them for degradation; and micro RNA (miRNA), annealing mainly to the 3’ UTR of certain transcripts and inhibiting their translation (Atayde, Tschudi and Ullu, 2011; Kolev, Tschudi and Ullu, 2011). In T. brucei miRNA have been predicted but not detected though (Mallick, Ghosh and Chakrabarti, 2008; Atayde, Tschudi and Ullu, 2011). In addition, metazoan also have a third type: PIWI-interacting small RNAs (piRNAs) (Siomi et al., 2011).

The RNAi pathway in Trypanosoma brucei has been reviewed in the literature (Atayde, Tschudi and Ullu, 2011; Kolev, Tschudi and Ullu, 2011). It involves two Dicer-like proteins: TbDCL1 (cytoplasmic) and TbDCL2 (nuclear) (Shi, Tschudi

and Ullu, 2006; Patrick et al., 2009). Both have RNase III activity and nuclease- cleave double stranded RNA (dsRNA) into ssRNA. TbDCL1 activity is assisted by cofactor TbRIF5 (Barnes et al., 2012). The resulting 20-30 nucleotide dsRNA fragments are protected from nucleases through methylation in their 3’ end by HEN1 activity (Horwich et al., 2007; Patrick et al., 2009). After separation of the two strands mediated by exonuclease TbRIF4 (Barnes et al., 2012), the targeted transcript is specifically bound by the complementary half of the ssRNA and loaded into another protein, AGO1 ‘slicer’. This assembly is called RNA-induced silencing complex (RISC) and is responsible for the ultimate mRNA cleavage that limits gene expression (Durand-Dubief and Bastin, 2003; Shi et al., 2004). The siRNA pathway has several endogenous functions: maintenance of genome integrity through transposon and retrotransposon silencing; post-transcriptional regulation; defence against viral invasion; chromosome segregation; and

heterochromatin formation. The RNAi pathway has also been exploited as a reverse genetics tool, becoming a real breakthrough in the post genomic era. Genetic manipulation enabled the exploitation of RNAi machinery to selectively target genes for transcript depletion. If their role was unknown, observation of the resulting phenotypes permitted examination of their putative functions. If a gene’s role had been already identified, RNAi could help understanding of how its absence or lack of activity affected other factors in the cell (Atayde, Tschudi and Ullu, 2011; Kolev, Tschudi and Ullu, 2011).

Unlike other Eukaryotes, T. brucei lacks RNA-dependent RNA polymerase (RdRp), meaning it is unable to expand or preserve exogenous dsRNA after replication. Thus, the effect after transfection of dsRNA is only transient (Siomi and Siomi, 2009; Kolev, Tschudi and Ullu, 2011). Full exploitation of RNAi was therefore not possible until development of a system for inducible expression of dsRNA. This involved integration of a DNA template in a silent genomic locus, from which the desired dsRNA could be inducibly expressed under control of a strong promoter controlled by a tetracycline operator (Orth et al., 2000; Shi et al., 2000). Since then, two different dsRNA expression strategies have been used: the ‘stem- loop’, and the use of ‘opposed promoters’. In the first system, the DNA sequence integrates in ‘sense’ and is followed by the complementary sequence in

stranded hairpin upon transcription by self-annealing of the RNA transcript. In the second approach, the desired RNAi target integrates in the genome flanked by opposed promoters. After transcription, complementary strands will result that anneal, producing the expected dsRNA. Both strategies were compared, showing stem-loop to have a better efficiency for mRNA depletion (Durand- Dubief, Kohl and Bastin, 2003). For the Tet-operator to be effective, cell lines need to be modified to express the tetracycline repressor from Escherichia coli, which blocks transcription of the gene located downstream of the promoter until displaced by tetracycline (Wirtz and Clayton, 1995).

Many variants of these systems, combining different genomic loci and promoters, have been applied to T. brucei research. The most commonly used has been the EP/GPEET procyclin promoter; however, this is down-regulated in mammal- infective forms. In bloodstream forms one of the most widely used promoters is T7, which requires parasites to be modified to express the T7 RNA polymerase (Wirtz et al., 1999). High levels of expression of this polymerase are toxic for trypanosomes. Therefore, weak promoters must be used in order to achieve balanced levels between efficacy and toxicity. RRNA promoters have been proposed instead as a robust endogenous alternative that can be used in T.

brucei regardless the life stage and permitting performance of differentiation

studies (Alsford et al., 2005).

Among the silent loci most successfully used to target inducible expression cassettes are the rRNA spacers. However, the T. brucei genome contains nine rRNA loci, each providing different transcription levels. After targeting a GFP reporter with the hygromicin resistance cassette (HYG), the best expressing locus was selected. Then, GFP was replaced with a puromycin resistance gene (PAC), while leaving HYG with only its 3´end as a non-functional tag. Such a strategy enabled accurate targeting of any RNAi construct to the tagged RRNA locus by engineering it to contain the missing 5´end of HYG. After successful homologous recombination, RNAi clones recover hygromycin resistance, while normally losing PAC (Alsford et al., 2005).