Selected candidate gene Pqbp1 is cap-independently translated in vitro

Out of 17 candidates that were tested for internal translation initiation in bicistronic reporter assays, Pqbp1 was the only one that passed the siRNA-based validation in both mESCs and HEK293 cells. Further, in mESCs Pqbp1 displayed both potential IRES activity which was as strong as EMCV IRES and potential CITE activity that substantially enhanced translation of the dependent reporter within a bicistronic reporter construct. To further determine the cap-dependency of Pqbp1 we generated differentially capped monocistronic reporters that carry

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either an ordinary m⁷Gppp-cap or a non-functional Appp-cap analogue. The cap-analogue itself is not recognized by the translational machinery i.e. it does not recruit ribosomes, but it protects the RNA from degradation. By comparing translational rates of m⁷Gppp-capped and Appp-capped RNAs one can distinguish the contribution of cap-dependent and cap-independent translation to the overall translational output of the transcript.

We performed in vitro translation assays using the differentially capped RNA reporters in a cell-free Drosophila embryo system. While the expression of the RLuc control reporter was widely reduced when m⁷Gppp-cap was replaced with cap analogue, CrPV IRES-, HCV IRES- and Pqbp1-mediated RLuc expression was unimpaired (Fig. 12A). The CrPV IRES reporter was even 3-fold more efficiently translated when it was Appp-capped, while Pqbp1- and HCV-mediated translation was only slightly increased (Fig. 12B). These results show that the contribution of m⁷Gppp-cap to Pqbp1 translation is low and that in vitro cap-independent translation of Pqbp1 is as efficient as cap-dependent translation.

However, translation levels of Pqbp1 and HCV IRES reporters were an order of magnitude lower than translation levels of the RLuc control reporter missing a 5’UTR (Fig. 12B).

This indicates that expression of both sequences is rather inefficient in the Drosophila system, probably due to inadequate concentration, absence or differential evolutionary conservation of specifically required initiation factors compared to mammals. Moreover, it is possible that the in vitro synthesized reporters lack RNA modifications that contribute to translation efficiency.

We went on to investigate how cap-independent translation of Pqbp1 behaves in vitro during cap-dependent translation inhibition. Therefore, we added the m⁷Gppp-cap in trans to the translation system. Excess of the cap-structure increases the competition between capped mRNAs in recruiting the translational machinery so that the m⁷Gppp-cap itself is of no translational advantage anymore and cap-independent translation initiation is more efficient.

For the m⁷Gppp-capped RLuc control reporter, translation is severely decreased by addition of the cap in trans (Fig. 12C). In contrast, m⁷Gppp-capped Pqbp1 reporter expression is even slightly increased after addition of the cap (Fig. 12C). While under normal conditions Pqbp1 reporter expression is an order of magnitude lower than RLuc control reporter expression, it slightly exceeds RLuc reporter expression under m⁷Gppp-induced cap-dependent translation inhibition. These results suggest that under specific conditions in which cap-dependent translation is diminished, cap-incap-dependent translation of Pqbp1 is sufficient to compete with repressed cap-dependent mRNAs at least in vitro.

The viral m⁷Gppp-HCV IRES behaves like Pqbp1 as it is expressed at similar levels and as its expression is stable during translation inhibition by excess m⁷Gppp (Fig. 12C). The m⁷ Gppp-CrPV IRES instead is two orders of magnitude more efficiently translated than Pqbp1, however,

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after addition of cap in trans, CrPV IRES translation reduced remarkably (Fig. 12C). Anyhow, during inhibition of cap-dependent translation CrPV IRES expression is still the most efficient one in this system. Such a dramatic decrease in CrPV IRES reporter expression seems unexpected, but it was already shown elsewhere that m⁷Gppp-capped viral IRESs perform worse under m⁷Gppp-induced translation inhibition than their Appp-capped counterparts in vitro [67].

Fig. 12: Pqbp1 is cap-independently translated in vitro. (A) Drosophila cell-free in vitro translation assay comparing translation of monocistronic Pqbp1 reporters carrying either m⁷Gppp-cap or Appp-cap analogue. Renilla luciferase activity (RLuc) was normalized to Firefly luciferase activity (FLuc) of a

m⁷Gppp-capped monocistronic FLuc internal control reporter. Error bars represent standard deviation of three biological replicates. CrPV and HCV reporters were used as positive controls and RLuc reporter lacking a 5’ UTR was used as negative control. (B) Individual RLuc and FLuc translation levels of

m⁷Gppp-capped or Appp-capped reporters shown in (A). Error bars represent standard deviation of three biological replicates. (C) Drosophila cell-free in vitro translation assay testing m⁷Gppp-capped Pqbp1 reporter in the presence or absence of cap-dependent translation inhibitor (m⁷Gppp added in trans).

m⁷Gppp-capped monocistronic FLuc reporter was used as internal translation control (lower panel). Error bars represent standard deviation of three biological replicates. m⁷Gppp-capped CrPV and HCV reporters were used as positive controls and m⁷Gppp-capped RLuc reporter lacking a 5’ UTR was used as negative control.

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In summary, the in vitro translation experiments show that Pqbp1 exhibits low cap-dependency and that cap-independent translation of Pqbp1 yields meaningful levels of protein expression when compared to cap-dependent and HCV IRES translation during inhibition of cap-dependent translation. Nevertheless, Pqbp1 translation is far less efficient as CrPV IRES-mediated translation in the Drosophila system, which might be explained by the fact that CrPV is specialized on insect infection whereas Pqbp1 was originally identified in mouse and HCV only infects humans and chimpanzees [873]. Thus, it is recommended to confirm cap-independent translation of Pqbp1 in a mammalian cell system, either in vitro or even better in vivo by RNA transfection.

After demonstrating that Pqbp1 can efficiently promote cap-independent translation, it remains to be clarified how efficiently Pqbp1 mediates 5’end-independent and internal translation initiation. In vivo assays in mESCs suggest that Pqbp1 can boost cap-dependent translation when located downstream of the ORF. This resembles the working mechanism of plant viral CITEs that enhance translation initiation from their location in the 3’UTR but can also function when placed at the 5’end [874]. CITE-like translation is 5’end-dependent with ribosomes scanning the 5’UTR from 5’ to 3’ direction. By inserting stable hairpins at the very 5’end of bicistronic or monocistronic Pqbp1 reporters it can be determined how much Pqbp1 translation initiation relies on a free 5’end. Further, hairpins in bicistronic reporters can reveal whether potential internal translation initiation of Pqbp1 is dependent on translation of the upstream cistron. Ultimately, it is recommended to perform the assays by RNA transfection of reporters to complement the siRNA-based control experiments which already suggested that Pqbp1 is free of cryptic splice sites or cryptic promoters causing artefacts.