Algoritmo de codificación

The observation that She2p can bind to unrelated stem-loop containing RNAs with significant affinity raised the question how She2p selectively recognizes its mRNA targets in the nucleus. Stem-loops are abundant secondary structures in any kind of RNAs (Svoboda and Di Cara, 2006). Thus, She2p could in principle bind to a number of mRNAs, tRNAs, or rRNAs in the nucleus, translocate them to the cytoplasm and incorporate them into the translocation complex. The only modest difference in affinity of She2p to specific and non-specific RNAs is unlikely to explain how She2p discriminates between bud-localizing mRNA and non-specific RNA.

One possibility is that She2p selects bud-localizing RNAs by recognizing conserved sequence motifs of 3 to 9 nucleotides in length within the zipcode, as it was recently proposed by Jambhekar et al. 2005 and Olivier et al., 2005. To test if She2p interacts with these short motifs alone, electrophoretic mobility shift assays (EMSA) were performed. Wild-type She2p was incubated with the respective radiolabeled RNA oligonucleotide and the RNA:protein complexes were separated from unbound RNA in a native polyacrylamide gel. As shown in Figure 12, She2p was able to bind to each of the short sequence elements, but the interaction was barely visible in EMSA experiments. Furthermore, a significant amount of unbound RNA was noticed, although a 2000-fold molar excess of protein was used in the individual reactions. This observation indicates that the monitored She2p:RNA association might be a rather transient interaction.

Figure 11: She2p dimerization is indispensable for RNA binding.The cartoons in a) and b) highlight in red serine 120 in the dimer interface that was mutated to tyrosine. a) shows She2p from the front and b) from the top. c) The mutant protein She2p-S120Y neither binds to any ASH1 zipcode nor to the stem-loop containing RNAs HIV-1 TAR and U1snRNA. The graph shows a representative plot of the relative bound E3-RNA fraction by She2p-S120Y compared to the RNA fraction that is bound by wild-type She2p. No dissociation constant of She2p-S120Y to any RNA could be determined (maximum protein concentration measured: 12 µM).

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In order to quantify the identified interactions and to analyze their stability, I performed SPR experiments using a Biacore 3000 system (see also chapter 2.1.6). RNA oligonucleotides (9- mer, 6-mer, 3-mer) with a 5’ biotin tag were immobilized on a streptavidin-coated chip surface and She2p was floated over the surface. If any interaction occurs, an increase in the response signal can be monitored. Analysis of the obtained sensorgrams revealed a fast association and dissociation of the complex (Figure 13 a). From the measured response curves no on-rates (K_on) or off-rates (K_off) could be derived in order to calculate the equilibrium-dissociation constant KD (KD= Koff/ Kon). However, the binding constant was calculated from steady state response signals of multiple injections with increasing protein concentrations by plotting the response signals at the equilibrium against the respective protein concentration. The experiments revealed that She2p bound to the 9-mer motif and the 6-mer motif with a binding constant of 18.6 µM and 20.7 µM, respectively (Figure 13 a-c). In contrast, no detectable binding was observed to the 3-mer motif (CGA) up to a protein concentration of 200 µM (Figure 13 c). I cannot exclude the possibility that the 3-mer RNA failed to bind due to its close proximity to the dextran matrix of the chip. In comparison to She2p’s affinity to “full- length” zipcodes (chapter 2.2.1), the binding constants measured to these short RNA sequences are significantly weaker. These findings indicate that short RNA motifs alone are unable to mediate specific She2p binding. However, it cannot be excluded that they act in combination with other nucleic acid features like secondary- and tertiary-structure elements to achieve specific zipcode recognition by She2p.

To test whether the combination of secondary-structure elements and an RNA-consensus motif results in a zipcode element, a rather simple approach was chosen: nucleotides in the

Figure 12: She2p binding to short RNA-consensus sequences can be detected. Electrophoretic mobility shift assays using a native 7 % polyacrylamide gel revealed binding of wild-type She2p to a 3-mer, 6-mer, 7-mer, and 9-mer RNA oligonucleotide (indicated by an asterisk). In the experiment, a 2000- fold molar excess of protein was used. However, a large amount of unbound RNA was detected, which might reflect a transient interaction.

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upper region of the HIV-1 TAR stem loop were replaced by the conserved 6-mer motif (CGACGA) in a way that preserved the predicted secondary structure. If the RNA motif would function in combination with a simple secondary structure, an affinity would be expected that is comparable to the affinity to zipcodes. As a control, the RNA motif was inserted in antisense orientation (GCUGCA), for which no increase in affinity would be expected. Secondary-structure predictions of both “artificial” zipcodes are shown in Figure A1 in the appendix. RNA filter-binding experiments revealed that wild-type She2p binds with similar affinity to both “artificial” zipcodes (KD=1.2 µM ± 0.2 µM and KD=1.0 µM ± 0.2 µM, respectively) as to the “wild-type” HIV-1 TAR RNA (compare Figure 13 c with Figure 10 a). This finding indicates that the nature of a zipcode is much more complex and cannot be simply generated by combining a conserved RNA motif with secondary-structure elements.