Complementarity between the mRNA 5 ' untranslated region and 18S ribosomalRNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome -binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for tran slation initiation in eukaryotes. Here, we used an antisense RNA approach i n vivo to identify the regions of 18S rRNA that might interact with the mRN A 5' untranslated region (5' UTR). Various fragments covering the entire mo use 18S rRNA gene were cloned 5' of a caf reporter gene in a eukaryotic vec tor, and translation products were analyzed after transient expression in h uman cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of th e downstream open reading frame (ORF). When translation inhibition is obser ved, reduction of the size of the complementary sequence to less than 200 n t alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory eff ect. Sucrose gradient fractionation of polysomes reveals that mRNAs contain ing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initia tion complexes. Our results support an mRNA-rRNA base pairing to explain th e translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.