Establishment of 5’-3’ interactions in mRNA independent of a continuous ribose-phosphate backbone

Abstract

The functions of eukaryotic mRNAs are characterized by intramolecular interactions between their 5' and 3' ends. Here, we have addressed the question whether such 5'-3' interactions are established by diffusion-controlled encounter of the ends 'through solution' or by some type of scanning along the RNA backbone. For this purpose, we used an in vitro translation system derived from Drosophila embryo extract that displays two types of 5'-3' interactions: cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug Recognition Elements (SREs) in the 3' UTR. Chimeric RNAs were constructed in which a luciferase open reading frame was separated from SREs and the poly(A) tail by a protein linker. Stimulation of translation by the poly(A) tail was fully functional with such RNAs even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between open reading frame and poly(A) segment. The stimulatory effect of the poly(A) tail also became weaker with increasing distance between the 5’ end and the poly(A) segment. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same RNA constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the RNA backbone is excluded as a mechanism for repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting the possibility that the contact between the SREs in the 3' UTR and the 5' end of the RNA is established in a manner that differs from the contact between poly(A) tail and the cap.