Biochemical and functional analysis of a 9-nt RNA sequence that affects translation efficiency in eukaryotic cells

Abstract

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 40S 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 18S 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 (Ϸ8-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.I n earlier studies, we identified cis-acting sequences within the 5Ј leaders of the mRNAs for the homeodomain protein Gtx and the cold-stress-induced protein Rbm3 that appeared to function as internal ribosome entry sites (IRESs). When tested in isolation these relatively short sequences enhanced the translation of a second cistron in a dicistronic mRNA (1, 2). In addition, we found that RNA sequences with IRES activity could be selected from libraries of random 9-to 18-nt sequences (3, 4). Although in most cases, IRES activity mediated by an individual IRES element was relatively weak, it could be greatly enhanced when multiple copies of some of the individual IRES elements were linked together in tandem (1, 3).Synthetic IRESs based on the 9-nt Gtx IRES module have proven to be particularly useful in various vector systems for generating high levels of expression from multicistronic mRNAs (e.g., refs. 5-7). Moreover, they are shorter and are more active than viral IRESs, such as the encephalomyocarditis virus IRES, that are typically used for these applications, and synthetic IRES activity can be enhanced or diminished by increasing or decreasing the number of IRES modules.The question arises whether, in addition to enhancing translation in a dicistronic context, such constructs would also enhance translation in a monocistronic context, i.e., in the 5Ј leader of a reporter mRNA. There are a number of reasons p...

Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA

“…Such multiple Gtx elements were used to increase the signal͞noise ratio as in our earlier studies (3,12,18). On comparing this construct with one containing an uAUG (p2ϩuAUG), it appears that Ϸ16% of ribosomes bypassed the uAUG.…”

“…Photinus luciferase reporter constructs were based on plasmids pGL3c 5Ј multiple cloning site and pYESFFlucH (3,12). Plasmids encoding yeast and mouseyeast hybrid 18S rRNAs (pNOY353, pVM1, and pVM4, respectively) were identical to those used in our previous studies (3).…”

“…Transfection of mouse N2a cells and transformation of yeast strain NOY908, reporter gene assays, and total RNA isolations were performed as described in our studies (3,12). Translation efficiencies are expressed as luciferase activities in raw light units normalized for luciferase mRNA levels.…”

“…To test the notion that mRNA-rRNA base pairing mediates shunting, we used a sequence element from the 5Ј leader of the Gtx homeodomain mRNA. In earlier studies, we showed that this element functioned as a binding site for 40S ribosomal subunits and that it enhanced translation by a mechanism that involved base pairing to a complementary segment of 18S rRNA (3,12). We now show that 40S ribosomal subunits can shunt across an upstream AUG or stable hairpin structure using Gtx elements as shunt donor and acceptor sites (13).…”

A Small Stem Loop Element Directs Internal Initiation of the URE2 Internal Ribosome Entry Site in Saccharomyces cerevisiae

“…Some IRES elements have been shown to be as small as 22 nucleotides (24). There is also a report of an element that interacts with ribosomal RNA to recruit the ribosome which is only 9 nucleotides in length (25). On the other end of the spectrum, the minimal sequence for functionality of some IRES elements have been reported to be larger than 300 nucleotides.…”