Análisis estructural y funcional del IRES de Picornavirus

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Centro de Biología Molecular Severo Ochoa. Fecha de lectura: 22-07-2010The IRES element of foot-and-mouth disease virus (FMDV), a picornavirus that causes a devastating disease worldwide, is organized in five structural domains. The distal domains are involved in RNA-protein interactions; in contrast, only a few host factors interact with the central domain, termed 3. Domain 3 contains several conserved motifs (namely, GNRA, RAAA and a C-rich bulge) and it is able to establish long-range RNA-RNA interactions. RNA structure is tightly linked to biological function. Although RNA probing has provided important insights into the secondary structure of this region, little is known about its three-dimensional structure. Thus, to understand the mechanism of IRES-dependent translation it is crucial to determine the arrangement of the stem-loops shaping up its structural organization. With the aim to define its structural organization, we have combined functional analysis, phylogenetic conservation and RNA probing. The genetic variability of RNAs viruses provides the best evidence of the structural requirements, as nearly all possible base–pair substitutions generating an RNA structure compatible with IRES function are selected in nature. Here, we have performed a comparative analysis of 183 sequences of FMDV representing all seven serotypes. The data revealed highly conserved motifs, indicating functional constrains for an RNA region endowed with the capacity to drive internal initiation of translation. Previous work has shown that a structural motif within the FMDV IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in culture cells, leading to inhibition of translation. This effect was dependent on the expression of the active ribozyme subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5´-end-dependent translation of the RNase P target RNAs. The results obtained from mutational analysis of conserved motifs within domain 3 of the FMDV IRES led to propose that this domain instructs the functional conformation of the whole IRES element. Here, we have used two independent approaches to assess the impact of global RNA structure on IRES activity. First, SHAPE reactivity revealed the self-folding capacity of the central domain, and provided new information regarding enhanced reactivity of specific residues located in conserved motifs. Second, comparison of single nucleotide reactivity with global RNA accessibility measured in oligonucleotide antisense microarrays revealed that SHAPE reactive nucleotides corresponding to the GNRA motif were not accessible to their respective target oligos in the microarray. This differential response, which was observed irrespectively of using the central domain alone or the entire IRES transcript, could be interpreted as the result of tertiary interactions. The GNRA motif dictates the organization and stability of domain 3. In this study we have confirmed that this effect is mediated by the interaction with distant residues. The study of a single nucleotide substitution of the GNRA tetraloop led to a strong RNA reorganization, affecting several nts away in the secondary structure of domain 3. In addition, we have observed that the RAAA motif contributes to the maintenance of RNA structure, as its disruption leads to rearrangement of the IRES structural organization. Terminal loops with a GNRA consensus sequence are a prominent feature of self-assembling RNA molecules. These interactions are specified by conserved set of nucleotides with the capacity to form non-canonical base-pairings or specific backbone conformations. Structural studies have demonstrated that specific receptors are recognized by GNRA tetraloops. While these interactions may have diverse sequence composition, they share similar structural organization, following rules that can be characterized. In this regard, it has been suggested that the last two bases of a GNRA loop could contact two consecutive purine bases in the minor groove of an A-RNA helix. In order to investigate tertiary interactions involving the GNRA tetraloops of the FMDV IRES we made several mutations (termed SR1-4) in the apical region of domain 3, composed of conserved C:G base-pairs. Nucleotide substitutions disrupting C:G base-pairs in the apical region impaired IRES activity. Structural analysis of SR mutants indicated gross changes in RNA structure affecting various regions (the substituted region, GNRA, loop 140 and 240 loop). Thus, the apical stem forms part of a functional binding site for the GNRA motif. Taken together, our results have been used to generate a model of a potential GNRA:receptor interaction, that involves at least four conserved motifs within the apical region of domain