Dissection of a rice OsMac1 mRNA 5’ UTR to uncover regulatory elements that are responsible for its efficient translation

Abstract

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5’ UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5’ 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5’ leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3’end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.