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We previously showed that a 9-nucleotide sequence from the 5′ leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5′ leaders, Gtx itself andFGF2(fibroblast growth factor 2). 2′-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinantmRNAscontaining the Gtx or FGF2 5′ leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5′ leaders was decreased by>50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx andFGF2 5′ leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellularmRNAsand describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells. [ABSTRACT FROM AUTHOR]

We previously identified an internal ribosome entry site (IRES) within the 5' leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5' leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5' leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 405 ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 185 rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced (≈8- fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA. [ABSTRACT FROM AUTHOR]