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NI Piriev, CK Yamashita, J Shih, NB Akhmedov, DB Farber; The Role of the 5' and 3' UTRs in the Regulation of PDE Translation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3653.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We demonstrated earlier that while the mRNA levels of photoreceptor-specific cGMP-phosphodiesterase (PDE) α and ß catalytic subunits are 1:5 in mouse retina, the ratio of the corresponding proteins is 1:1. We then showed that PDEα mRNA is translated more efficiently than PDEß mRNA, compensating in this way the differences in mRNA levels. In this study we analyzed the role that the PDEα and PDEß mRNA 5' and 3' UTRs play in determining the efficiency of protein synthesis. Methods: Equal amounts of in vitro synthesized RNAs were used for all in vitro translation studies. Resulting proteins were quantified by scintillation counting of the labeled bands separated by PAGE. Potential stem-loop structures of mRNAs were analyzed using the "RNA Secondary Structure Prediction Program" of Genebee Services. Results: Hybrid constructs containing different combinations of coding regions and 3' and 5' UTRs of PDEα and PDEß (there are two PDEß transcripts, one with a short and the other with a long 5' UTR) were prepared. The amount of protein synthesized using the hybrid construct containing PDEα 5' UTR and PDEß coding region and 3' UTR was about 5.5-fold lower than that produced by the native PDEα transcript. In contrast, placement of the PDEß short 5' UTR in front of the PDEα coding region and 3' UTR resulted in ∼2-, 8- and 10-fold greater protein synthesis than that produced by the native PDEα and PDEß with short or long 5' UTR, respectively. Placement of the PDEß long 5' UTR in front of the PDEα coding region and 3' UTR, also increased protein synthesis above the levels produced by the native mRNAs. Using other chimeric constructs we demonstrated that PDEα and PDEß 3' UTRs have a stimulatory effect on translation. However, the PDEα 3' UTR stimulatory effect was not observed when constructs contained the PDEα 5' UTR. We explain this by possible base pairing interactions between PDEα 5' and 3' UTRs. Conclusion: We have analyzed the effect of PDEα and PDEß 5' and 3' UTRs on translation and showed that both the short and long PDEß 5' UTRs lead to more protein synthesis than the PDEα 5' UTR. Although both the PDEα and PDEß 3' UTRs had a stimulatory effect on translation, it appears that 3' and 5' UTRs of PDEα could be involved in base pairing interaction reducing protein synthesis efficiency. No such interaction was observed with the PDEß 3' and 5' UTRs. Moreover, our results indicate that the PDEα and PDEß coding regions are involved in the differential expression of these subunits, with the former producing higher protein level than the latter.
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