Hipótesis especifica 1

The structural studies on eukaryotic Argonaute PAZ domains revealed that the PAZ domain provides a unique platform for the recognition of the two 3’-terminal nucleotides in single- stranded nucleic acids. A key feature of the binding is the burial of the terminal 3’-OH group. This binding mode provides new insights into the biological role of this domain within Dicer and Argonaute proteins, which constitute the cores of RNA silencing complexes.

In the initiation step of RNA-mediated gene silencing, the RNase III enzyme Dicer generates small double-stranded RNAs. Based on the established function of the Argonaute PAZ domain, a model of PAZ function in Dicer was proposed by Filipowicz and co-workers in a recent study addressing the function of human Dicer (Zhang et al., 2004). The authors suggest that the characteristic size (~20 bp) of the siRNAs resulting from Dicer cleavage may be linked to 3’-end recognition of the dsRNA substrate by the Dicer PAZ domain. In this model, the PAZ domain recognizes the 2 nt 3’-overhang generated by Drosha in the miRNA pathway, or the last cut made by Dicer in the RNAi pathway. PAZ and RNase III domains of Dicer are spaced such that when the 3’-overhang is bound, they act as a ruler to position the cleavage site exactly 22 nt away from the 3’-end. This model is consistent with the known

cleavage at the ends of dsRNA and pre-miRNAs, shortening the substrate consecutively by siRNA-sized segments, and not cutting internally, which would lead to the generation of multiple larger fragments (Zhang et al., 2002; Zhang et al., 2004; Vermeulen et al., 2005). In- line with this model, both the PAZ and dsRBD domains are required for full Dicer activity. Intact Dicer can slowly process blunt-ended dsRNA, however deletion of the dsRBD makes

cleavage of long dsRNA, the Dicer-2-R2D2-siRNA rnary complex is an assembly intermediate in the formation of RISC (Pham et al., 2004; Tomari et al., 2004b) that is proposed to recruit Ago2 directly to the siRNA. This interaction uplex is hereb d from the initiator complex into the RISC effector complex, as it was Dicer more dependent on substrates with 3’-overhangs (Zhang et al., 2004). This suggests that the dsRBD contributes to non-specific binding of incoming substrates and potentially allows Dicer to cleave long dsRNA substrates processively. As no experimental structural data is available for the PAZ domain of Dicer proteins, the exact details of the catalytic mechanism and the role of the PAZ domain must await the structure of a Dicer-dsRNA complex.

In Drosophila, Dicer-2 is stably associated with the dsRBD containing protein R2D2 (Liu et al., 2003), and they are together required for cleavage of dsRNA and for RISC assembly (Lee et al., 2004b; Pham et al., 2004). After

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might be mediated directly by Ago2 and Dicer-2 (Tahbaz et al., 2004). The siRNA d y transferre

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demonstrated recently (Matranga et al., 2005). The results on the PAZ domain of Argonaute proteins presented in this thesis show that it specifically recognizes the 2 nt 3’-overhang of siRNAs. This suggests that the PAZ domain is involved in receiving the siRNA from Dicer and to facilitate its incorporation into RISC.

The role of the PAZ domain in the subsequent catalytic cycle of RISC can be envisioned as follows: The PAZ domain of Ago2 binds the 3’-end of the strand that is considered the guide strand of the siRNA duplex. At the same time, the 5’-end of the guide strand is docked in the phosphate binding pocket of the Ago2 Piwi domain (see chapter 1.2.4). The decision which strand of the siRNA duplex functions as guide strand is determined by the orientation of the siRNA in the RISC loading complex. The passenger strand would then be cleaved by Ago2 and the resulting fragments released (Matranga et al., 2005; Rand et al., 2005). This release might be facilitated by an ATP-dependent cofactor, like the release of the products of target mRNA cleavage which was shown to be stimulated by ATP (Haley & Zamore, 2004). The resulting RISC contains Ago2 with the single-stranded guide RNA bound. This ‘mature’ RISC provides the sequence-specific binding site for the complementary mRNA. The mRNA can then base pair with the guide strand and is cleaved by the endonucleolytic activity

constituted by the Piwi domain of the Argonaute protein (see chapter 1.2.3). The reaction cycle is completed with the release of the cleavage products.

It is not established yet that the PAZ domain interacts with the 3’-end of double-stranded or single-stranded siRNAs in vivo. Experiments testing this are possible and will be done certainly in the future. A strong indication that the PAZ domain is important for RNAi function in vivo is provided by results of a genetic screen in C. elegans (Tabara et al., 1999). Worms deficient in RNAi were isolated in which only an absolutely conserved glutamate in the PAZ domain of Rde-1 is mutated to lysine. The structure of the Drosophila Ago2 PAZ domain suggests that this mutation destabilizes the fold, which most probably also disrupts the structural integrity of the PAZ domain in vivo.

Despite the diversity of functions attributed to the different Argonaute-associated complexes (see chapter 1.2.1), a common feature of these complexes is the use of non-coding RNAs to select their targets in a sequence-specific manner. These non-coding RNAs (siRNAs or microRNAs) have the specific features of RNAs processed by the RNase III enzyme Dicer in common, i.e. a 19-22 nucleotide double-stranded RNA body with two nucleotide 3’- overhangs and 5’-monophosphate groups. This raises the question of how siRNAs or miRNAs are discriminated from other cellular RNAs and specifically incorporated into RISCs. With the binding specificity of the PAZ domain of Argonaute proteins, it is likely that it provides a contribution to the specific recognition of the 2 nt 3’-overhang feature of siRNAs. It would then act as a selectivity filter in RISCs, allowing only small RNAs processed by Dicer to enter the effector complex and excluding all other RNAs that are present in the cell. Without such a filter, all double-stranded RNA that is present in a cell could enter the RNAi pathway. This selectivity is very important, as the incorporation of unwanted dsRNA would lead to the down-regulation of the expression of complementary genes.