Bicistronic reporter assays in human embryonic kidney and mouse embryonic stem

Bicistronic reporters were cloned for 17 of the 26 selected candidate genes. For the remaining eight candidate genes amplification of the 5’UTR was unsuccessful. The candidate reporters were tested in mESCs as well as in HEK293 cells to determine if potential IRES elements are conserved in different cell types. Further, candidate reporter plasmids were co-transfected with 4E-BP1(4Ala) to test candidate’s IRES activity as well as their stimulation under conditions where cap-dependent translation is inhibited.

In mESCs, dual luciferase reporter assays revealed high translational activity for two tested candidates, Fam96b and Riok1, which was ~3-fold or ~12-fold higher than translational activity of EMCV (Fig. 8A). However, after 4E-BP1(4Ala) overexpression, ratios of candidate reporter expression decreased by half whereas the ratio of EMCV reporter expression increased by half. In general, translation initiation on IRES elements is triggered in the context of

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diminished cap-dependent translation. Since IRESs are unaffected by the mechanism of inhibition they have an advantage over the cap structure in recruiting the translational machinery.

Components of the translational machinery are in turn readily accessible, as ribosomes are less frequently engaged in translation. Hence, less of them are bound to mRNA and more are freely available in the cytoplasm. The decrease in Fam96b- and Riok1-mediated translation in this setting is indicating that their mode of translation initiation is rather unlikely to be an internal one.

The other 15 candidates showed lower translational activity ranging from the same order of magnitude to an order of magnitude below the translational activity of EMCV (Fig. 8A).

Further, the ratio of RLuc/FLuc translation from candidate reporters slightly decreased or did not change upon 4E-BP1(4Ala) induced inhibition of cap-dependent translation except for Pqbp1, whose RLuc/FLuc translation ratio slightly but insignificantly increased (Fig. 8A).

To gain better insights into translational abilities of the tested candidates, absolute values of RLuc and FLuc reporter expression were compared to each other. The absolute values confirmed that an unchanged ratio of most candidate reporters was the result of an equal decrease in cap-dependent FLuc translation and candidate-mediated RLuc translation after 4E-BP1(4Ala) overexpression (Fig. 9A). This indicates that most of the candidates don’t own IRES activity.

Strikingly, absolute values also revealed that Pqbp1 does not only promote efficient translation of the downstream RLuc ORF but also stimulates translation of the upstream FLuc ORF of bicistronic reporters (Fig. 9A). Further, the slight increase in RLuc/FLuc expression ratio was caused by a relatively less decrease in RLuc expression compared to FLuc expression and did not result from stimulated internal translation initiation. Hence, Pqbp1 is rather behaving like a CITE in the bicistronic setting where it enhances translation of both cap-dependent and potentially internally mediated translation under physiological conditions but does not possess strong IRES activity when cap-dependent translation is repressed.

The bicistronic reporters were tested as well in HEK293 cells. In this cell line all reporters except for one showed very low translational activity ranging from one to two orders of magnitude below EMCV-mediated reporter expression (Fig. 8B). All of these lowly translated candidate reporters displayed a decrease in RLuc/FLuc expression ratios after 4E-BP1(4Ala) co-transfection, suggesting that the tested candidate sequences are unable to mediate internal translation initiation in HEK293 cells. This was also confirmed by absolute reporter expression levels (Fig. 9B).

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Fig. 8: Candidate genes show activity in bicistronic reporter assays. (A, B) Dual luciferase reporter assays in wt mESCs (A) and HEK293 cells (B) transfected with bicistronic reporters containing 5’UTR sequences of candidate genes in between two luciferase cistrons. Cap-dependent Firefly luciferase (FLuc) translation and IRES mediated Renilla luciferase (RLuc) translation levels were determined 24 h after transfection with and without 4E-BP1(4Ala) co-transfection.

Error bars represent standard deviation of three biological replicates. EMCV IRES reporter and non-functional EMCV* IRES reporter were used as controls. (C, D) RNAi based validation of bicistronic candidate reporters in dual luciferase assays in wt mESCs (C) and HEK293 cells (D). Cells were co-transfected with bicistronic reporter vectors and siRNAs targeting FLuc (+ siRNA) or non-targeting control siRNAs (- siRNA). FLuc and RLuc activities were determined 24 h post transfection.

Error bars represent standard deviation of three biological replicates. Bicistronic EMCV IRES reporter and monocistronic FLuc and RLuc reporters were used as controls.

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However, one of the candidates performed better in the bicistronic assay than the others:

Pqbp1 displayed high translational activity that was ~2-fold as high as the translational activity of EMCV (Fig. 8B). Upon 4E-BP1(4Ala) co-expression, Pqbp1-mediated RLuc/FLuc expression level remained high, but did not increase, which contrasts with EMCV-mediated reporter expression that increased 1.8-fold. This suggests, that the translational activity of Pqbp1 could be of physiological relevance in HEK291 cells. In contrast to mESC assays, Pqbp1 did not enhance translation of the upstream FLuc reporter in bicistronic reporters in HEK293 cells (Fig. 9B).

This indicates that Pqbp1 was not acting as a CITE in HEK293 cells and although Pqbp1 was not acting like a bona fide IRES element either, it mediated potential internal translation initiation with similar efficiency as EMCV IRES.

In summary, bicistronic reporter assays could detect only low levels of potential internal translational activity that was around background expression levels for most tested candidates in both mESC cells and HEK293 cells. As EMCV IRES was also just weakly translated in mESCs (an order of magnitude lower than in HEK293 cells), candidates performed comparably better in mESCs than in HEK293 cells although their ability to mediate potential internal translation initiation was similarly low in both cell types. In order to confirm that potential internal translation initiation was mediated by an IRES and not caused by artefacts of bicistronic reporters that can arise during transcription, we performed siRNA-based control experiments.

Thereby the validation of Fam96b, Pqbp1 and Riok1 was of special interest, as the three of them promoted notable levels of potential internal translation initiation that were higher or in the range of EMCV IRES-mediated translation.

3.4.2 siRNA-based validation of bicistronic reporter constructs in human embryonic kidney and