Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry

The internal ribosome entry sites (IRES), IRESCP,148CR IRESMP,75,(CR) and IRE MP, precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus-encephalomyocarditis virus-in plant, yeast, and HeLa cells identified the 148-nt IRESCP,148CR as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRESCP,148CR are responsible for these features. On the basis oil' those findings, we designed artificial PARS-containing elements and showed that they, too, promote internal translation from dicistronic transcripts in vitro, in tobacco protoplasts and in HeLa cells. The maximum IRES activity was obtained from multiple copies of either (A)(4)G(A)(2)(G)(2) or G(A)(2-5) SCR as contained in IRESCP,148CR. Remarkably, even homopolymeric poly(A) was moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a database search for existing PARS sequences in T-untranslated regions (5'UTR) of genes in tobacco genome allowed the easy identification of a number of IRES candidates, in particular in the 5'UTR of the gene encoding Nicotiana tabacum heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5'UTR of NtHSF1 turned out to be an IRES element active in vitro, in plant protoplasts and HeLa cells. We predict that PARS elements, when found in other mRNAs, will show a similar activity.