pubmed.ncbi.nlm.nih.gov

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

  • ️Tue Jan 01 2002

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

Yuri L Dorokhov et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The internal ribosome entry sites (IRES), IRES(CP,148)(CR) and IRES(MP,75)(CR), 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 IRES(CP,148)(CR) as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRES(CP,148)(CR) are responsible for these features. On the basis of 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) as contained in IRES(CP,148)(CR). 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 5'-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.

PubMed Disclaimer

Figures

Figure 1
Figure 1

IRES-mediated GUS gene expression in tobacco plants transgenic for dicistronic CP-IRES-GUS constructs. Five series of transgenic plants differing in IRES sequences were generated: (I) Negative control: vector-transformed plants; (II) positive control: plants transgenic for monocistronic GUS gene; (III–V) IRESformula image, IRESformula image, and IRESformula image elements, respectively, were used as intercistronic spacers. (A) Histochemical detection of GUS activity. (B) Northern blot of total RNA isolated from transgenic tobacco leaves probed with a GUS gene DNA probe. Positions of synthetic monocistronic (GUS) and dicistronic (CP-IRESformula image-GUS) RNA transcripts are marked by arrows. (C) Western blot analyses of the crTMV CP in transgenic tobacco leaves. The arrow indicates the position of crTMV CP. Arabic numerals (B, C) denote the number of the transgenic plant line used. Roman numerals denote transgenic plants transformed with different constructs indicated above. (D) IRESCR-mediated GUS activity expressed in two different transgenic lines (denoted by Arabic numerals). The relative GUS activity was normalized to the CP content measured by densitometry of the CP bands presented in C.

Figure 2
Figure 2

Cross-kingdom conservation of IRESformula image activity. Expression of the 3′-proximal GUS gene from dicistronic CP-IRES-GUS constructs in tobacco protoplasts (A), HeLa (B), yeast (C) cells and cell-free translation systems WGE (D), and RRL (E). The 72-nt synthetic GC-rich polylinker-derived (PL72) spacer (23) (A, B) and the 148-nt region upstream from start codon of the CP gene of TMV U1 (U1formula image, ref. 22) (C) were used as negative controls. GUS gene expression in HeLa (B) and yeast (C) cells transfected with animal cell or yeast cell promoter-based dicistronic constructs H-GFP-IRES-GUS and CP-IRES-GUS, respectively.

Figure 3
Figure 3

IRESformula image deletion analysis. (A) Simplified schematic representation of the IRESformula image structure (see ref. for details) and its deletion mutants. Letters indicate the sequences of the 32-nt polypurine tract PPT32 (which includes the 19-nt element PPT19) located upstream of the hairpin–loop structure and the 11-nt tract (PPT11) just upstream of the CP gene, respectively. Arabic numerals indicate the nucleotide positions in full-length crTMV genomic RNA (21). The arrow points to the position resulting in formation of two deletion mutants (Δ5′IREScp and Δ3′IREScp) described in ref. . The lines indicated by PPTformula image and PPTformula image correspond to the respective IRESformula image deletion mutants used in the present study. GUS gene expression by internal translation from dicistronic constructs in WGE (B) and tobacco protoplasts (C) under control of the intact IRESformula image and its deletion mutants (PPTformula image and PPTformula image). UIformula image sequence (22) was taken as a negative control.

Figure 4
Figure 4

Comparative dicistronic analysis of IRES activities of multiple G(A)3 modules and natural IRESs (IRESformula image and IRESEMCV) in WGE (A), tobacco protoplasts (B), and HeLa cells (C). Artificial sequences tested: (i) (PPT19)4 and (PPT19)8 representing the tandem repeats of four (76-nt) and eight (152-nt) copies of the 19-nt AAAAGAAGGAAAAAGAAGG sequence derived from PPT32 (see Fig. 3), respectively; (ii) the 64-nt (GAAA)16 sequence consisting of 16 G(A)3 elements; (iii) control U-rich sequence (GUUU)16; (iv) the control Emp × 4 sequence consisting of four copies of the U-rich CGUUUGCUUUUUGUAGUA element derived from another crTMV IRES (IRESformula image) and (v) the GCU-rich sequence (GCU-R) containing four copies of CGCGGGCG blocks linked via the 7-nt sequence UUUGUUU used as an additional negative control. (A) Analysis of proteins directed in WGE by dicistronic H-GFP-ICS-GUS T7 transcripts containing artificial sequences as the intercistronic spacer. Arrows indicate the position of GUS and GFP. (B and C) GUS gene expression in tobacco protoplasts (B) and HeLa (C) cells transfected with dicistronic GFP-IRES-GUS constructs containing different IRES sequences. “Mock” indicates that DNA-free solution was used for transfection.

Figure 5
Figure 5

Dicistronic analysis of IRES activity of the 5′-UTR of NtHSF-1 mRNA (5′UTR NtHSF) in RRL (A), tobacco protoplasts (B), and HeLa cells (C). Tested H-GFP-ICS-GUS RNA transcripts contained as intercistronic spacers the 453-nt 5′UTR of NtHSF-1 mRNA (5′UTR NtHSF) and other synthetic sequences indicated in the legend to Fig. 4.

Similar articles

Cited by

References

    1. Pain V M. Eur J Biochem. 1996;236:747–771. - PubMed
    1. Kozak M. Gene. 1999;234:187–208. - PubMed
    1. Dever T E. Trends Biol Sci. 1999;24:398–403. - PubMed
    1. Jackson R J. In: Translation Control of Gene Expression. Sonenberg N, Hershey J W B, Mathews W B, editors. Plainview, NY: Cold Spring Harbor Lab. Press; 2000. pp. 127–184.
    1. Gale M, Tan S-L, Katze M G. Microbiol Mol Biol Rev. 2000;64:239–280. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources