REFlex sensor performance

4.5.1 REFlex assay in vitro application

Using REFlex we were able to monitor assembly and disassembly of the EJC in solution. In vitro REFlex assay provides a fast and conveniently tool to test candidates or screen for factors stabilizing or destabilizing the EJC. Single molecule FRET studies on RNA helicase conformational change as performed on the helicase YxiN are shedding light on the switching behaviour and function of these proteins (Theissen et al. 2008).Our FRET sensor REFlex might be a useful complementation to biochemical and structural studies in gaining more insight into the eIF4AIII interactome and elucidating its function in RNA regulation. The design principle can be applied to other members of the RNA helicase family.

In particular, we verified that PYM is only able to bind MAGO-Y14 if it is incorporated in the EJC. Our data support the hypothesis that this is due to conformational changes of MAGO- Y14 and does not depend on the subcellular separation of free cytosolic PYM and nuclear MAGO-Y14 (Gehring, Lamprinaki, Kulozik, and Hentze 2009c). FRET monitoring by REFlex demonstrates that Imp13 is sufficient to induce the transition of eIF4AIII to the open state in the absence of PYM. It will be interesting to study the relevance of this finding in the cellular context, in particular if one considers that Imp13 has a higher affinity for MAGO compared to PYM (Budiman et al. 2009).

RNA binding of REFLex in the presence of EJC core components was found to be unspecific. In the absence of these EJC components, however, in minimal assays employing only REFLex and ATP, a higher selectivity for a given RNA target was found. While RNAs representing the 3’ UTRs of two mRNAs subject to EJC related NMD (Sauliere et al. 2010) proved efficient targets for REFLex, no FRET change could be seen when an unrelated intronless mRNA coding for the fluorescent protein mKO2 was used in concentrations that were efficient in promoting EJC assembly in the presence of MAGO-Y14 and Barentsz. This suggests a reduced RNA selectivity of eIF4AIII when situated within the EJC core, while free eIF4AIII apparently retains preference for some RNA motifs over others. Along these lines, free eIF4AIII was reported to preferentially bind certain RNA loop structures within mRNAs coding for selenoproteins (Budiman et al. 2009). However, there is also recent evidence for RNA sequence-specific deposition of the EJC (Sauliere et al. 2010). In view of our data this

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could be explained by an initial binding of preferred sites by free eIF4AIII, which is subsequently stabilized and complemented by the other components of the EJC. It will be interesting to repeat the REFlex specificity test with a nuclear extract supplement including splice factors.

4.5.2 REFlex application for cellular imaging

4.5.2.1 REFlex function in cells

REFlex is a genetically encoded FRET sensor that is potentially suitable to study eIF4AIII in its two different states in living cells. Assigning the subcellular FRET signals to either the eIF4AIII bound or unbound form would provide a powerful tool to get functional insights. REFlex was transiently expressed under the control of a CMV promoter in HEK 293T cells as well as neurons. Within the endogenous cellular context, EJC components are expressed at a specific ratio (Gehring, Lamprinaki, Kulozik, and Hentze 2009b). Consequently, cellular REFlex as well as endogenous EJC component expression levels will be crucial for the study of eIF4AIII function. Particularly, endogenous eIF4AIII replacement by REFlex using a knock- in strategy would ensure usual cellular protein levels.

As live cell application requires high reporter sensitivity, sensor engineering is required. REFlex performance has been improved by insertion of mutations that favor FP dimerization. Targeted mutagenesis of amino acids at the dimer interface (Kotera et al. 2010), random linker insertion or random mutagenesis provide further means to improve the dynamic range even further.

4.5.2.2 Future application of REFlex for cellular imaging

In Drosophila oocytes, localization of oskar mRNA to the posterior pole is dependent on the nuclear formation of an RNP including the EJC. Another prerequisite for proper oskar mRNA targeting is splicing of the first intron. In the absence of eIF4AIII, oskar mRNA is evenly distributed in the oocyte. Labeling of oskar mRNA and REFlex coexpression would allow to subcellularly determine the pool of eIF4AIII-bound oskar mRNA compared to the dissociated

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situation. In this way, REFlex serves as a tool to study the requirement of EJC binding for proper cellular targeting.

Figure 73 Oskar mRNA and the EJC in Drosophila oogenesis. (A)(B) Stages 3-5 and 10b of oogenesis. Oskar mRNA (green) oocyte autofluorescence (red) (A) wt oocytes with oskar mRNA posterior localization (B) Y14 oocytes with oskar mRNA random distribution (Hachet and Ephrussi 2001e) (C)

Antibody staining for eIF4AIII during oogenesis. Posterior region of a stage 9 egg chamber expressing GFP–Mago (Isabel M. Palacios, Gatfield, Daniel St Johnston, and Izaurralde 2004b).

REFlex expression in neurons shows a somatodendritic distribution with granular localization in dendrites (Figure 74). Giorgi et al. found that eIF4AIII is associated with neuronal mRNA granules and dendritic mRNAs (Giorgi, Yeo, et al. 2007c). eIF4AIII knockdown increases both synaptic strength and GLUR1 AMPA receptor abundance at synapses. The expression of some proteins including Arc, which is required for maintenance of LTP, is increased by eIF4AIII depletion. The EJC is supposed to mediate translation- dependent mRNA decay that might advantageously function as critical brakes for protein synthesis in neurons that are highly dependent on spatially and temporally restricted protein expression. The association of eIF4AIII with dendritic mRNAs suggests that they have

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not yet undergone translation (Maquat 2004). REFlex provides a tool to get further insight into the role of eIF4AIII and its target mRNAs in maintaining synaptic strength.

Figure 74 eIF4AIII in neurons. (A) FISH of Arc mRNA (red) and immunofluorescence of 4AIII (green) after BDNF treatment. Right panels: enlargement of indicated dendritic region. Arrows indicate examples of colocalization. scale bar 10 mm. (C Giorgi, GW Yeo, et al. 2007c). (B) Hippocampal neurons transiently expressing REFlex (left, middle, right: green) and dendritic RNP marker ZBP- mRFP (red, right).

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