A 9-nt Segment of a Cellular mRNA Can Function as an Internal Ribosome Entry Site (IRES) and When Present in Linked Multiple Copies Greatly Enhances IRES Activity

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragme...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-02, Vol.97 (4), p.1536-1541
Main Authors: Chappell, Stephen A., Edelman, Gerald M., Mauro, Vincent P.
Format: Article
Language:English
Subjects:
5' Untranslated Regions - genetics
Animals
Biochemistry
Biological Sciences
Cell Line
Cell lines
Cells
Encephalomyocarditis virus
Genes
Genes, Reporter
Genetic vectors
Gtx protein
Homeodomain Proteins - genetics
Humans
Messenger RNA
Mice
Neurons
Nucleotides
Oligoribonucleotides - genetics
Proteins
Rats
Ribonucleic acid
Ribosomes
Ribosomes - genetics
RNA
RNA, Messenger - genetics
RNA, Ribosomal, 18S - genetics
rRNA 18S
Sequence Deletion
Transfection
Untranslated regions
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Summary:This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.97.4.1536