Tailoring the switch from IRES-dependent to 5'-end-dependent translation with the RNase P ribozyme

Abstract

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5'-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.

FIGURE 1.

(A) Diagram of ribozyme-IRES constructs. Three versions of the RNase P ribozyme (PCC6803 rnpB wild type, the GG to CC mutation in helix P4, and the antisense orientation of the rnpB gene) were combined with three different sequences of the IRES element (the wild type, the GUAG mutation in the GNRA motif, and the CGCCC mutation in the RAAA motif). A schematic representation of the FMDV IRES structure with indication of the different domains, the location of the GNRA and RAAA motifs, and the RNase P cleavage site (depicted by an asterisk) mapped in vitro is represented at the top. CAT and luciferase indicate the reporter genes. (B) Determination of reporter RNA amount. Real-time RT-PCR analysis of reporter RNAs in cells transfected with the different versions of the ribozyme constructs. The amount of RNA corresponding to domain 3 (nucleotides 880–1022, relative to the +1 residue of the reporter RNA) or domains 4–5 (nucleotides 1007–1101) of the FMDV IRES was determined in duplicate assays of three independent experiments by RT-qPCR and made relative to the amounts of the 5′ sequence (nucleotides 4–147) present in the same samples. Error bars indicate the standard deviation. The Student's t-test was used for statistical calculation, and P values are indicated.

FIGURE 2.

Expression of the active rnpB ribozyme inhibits IRES activity. (A) Time course of reporter translation in response to expression of the rnpB ribozyme. The luciferase activity (measured as relative light units, RLU) of extracts prepared from cells transfected with the indicated constructs (0.33 μg/10⁵ cells) was determined over a period of 24 h and normalized to the concentration of protein in the extract. Values of luciferase activity/protein correspond to the average obtained from triplicate wells and performed in at least three independent assays. (B) Immunodetection of luciferase expression by Western blot. (C) Levels of inhibition of IRES-dependent translation by expression of the RNase P ribozyme. The luciferase activity determined at the indicated time in extracts of cells transfected with the ribozyme constructs was normalized to the value observed in the IRES construct set to 100%. Error bars correspond to the standard deviation.

FIGURE 3.

Insertion of structured RNA upstream of the IRES region does not affect IRES activity. (A) Time course of reporter translation efficiency in construct expressing the antisense sequence of the rnpB ribozyme upstream of the IRES region. Values correspond to the average of luciferase activity obtained from triplicate wells and performed in three independent assays. (B) Lack of inhibition of IRES-dependent translation by expression of the antisense sequence of rnpB. The luciferase activity determined at the indicated time in extracts of cells transfected with the asRz-IRES construct was normalized to the value observed in the IRES construct set to 100%. Error bars correspond to the standard deviation. (C) Expression of the antisense sequence of the rnpB gene does not induce RNA cleavage. RT-qPCR was used to determine the RNA amount corresponding to domains 3 and 4–5 of the IRES.

FIGURE 4.

RNase P recognition of the defective GUAG IRES leads to increased expression of the reporter. (A) Ribozyme-mediated cleavage of the reporter RNA. Real-time RT-qPCR analysis of reporter RNAs in cells transfected with the different versions of the ribozyme fused to the GUAG IRES. Data are expressed as in Fig. 1B. (B) Time course of reporter translation in response to expression of the rnpB ribozyme, fused to the IRES element carrying the single GUAG mutation in the GNRA motif. Data are represented as in Fig. 2A. (C) Changes in translation levels induced by expression of the RNase P ribozyme. Data are represented as in Fig. 2C.

FIGURE 5.

RNase P recognition of the defective CGCCC IRES induces an increase in reporter translation. (A) Ribozyme-mediated cleavage of the reporter RNA. Real-time RT-qPCR analysis of reporter RNAs in cells transfected with the different versions of the ribozyme fused to the CGCCC IRES. Data are expressed as in Fig. 1B. (B) Time course of reporter translation in response to expression of the rnpB ribozyme, fused to the IRES carrying a CGCCC sequence in the RAAA motif. Data are represented as in Fig. 2A. (C) Changes in reporter levels induced by expression of the RNase P ribozyme. Data are represented as in Fig. 2C.

FIGURE 6.

Levels of IRES-dependent translation in response to coexpression of the rnpB ribozyme with the Lb protease. (A) BHK-21 cells were transfected with the pBIC IRES construct (0.2 μg/10⁵ cells) in the presence or absence of the Lb-expression plasmid (0.033 μg/10⁵ cells). Extracts prepared at 4 and 8 hpt were used to measure CAT (IRES construct) or luciferase (all constructs). (B) Levels of luciferase activity found in the ribozyme-IRES constructs were made relative to those detected in the extract corresponding to the control IRES vector (54497300 RLU 4 hpt, 164576200 RLU 8 hpt), set to 100%.

FIGURE 7.

(A) Switch from IRES-dependent to 5′-end-dependent translation in defective IRES constructs in response to coexpression of the rnpB ribozyme with the Lb protease. BHK-21 cells were transfected with the ribozyme-GUAG IRES constructs in the presence or absence of the Lb-expression plasmid. Lysates collected 4 hpt were used to measure CAT (IRES construct) or luciferase (all constructs). Levels of luciferase activity measured in the ribozyme-IRES constructs were made relative to those detected in the extract corresponding to the control GUAG IRES vector (591200 RLU 4 hpt), set to 100%. (B) Primer extension analysis of the ribozyme cleavage sites. Total RNA isolated from cells transfected with Rz-IRES, RzP4-IRES, Rz-GUAG, or asRs-GUAG was subjected to RT extension with a 5′-end-labeled primer complementary to the IRES sequence. cDNA products were analyzed on denaturing 6% acrylamide gels in parallel with a DNA sequence prepared with the same primer. IRES nucleotides (numbered as in Serrano et al. 2007) are indicated on the left. Arrows denote the position of RT stops specifically detected in the Rz-IRES and Rz-GUAG RNA. Full-length products are show at the top. The relative intensity of RT stops observed in lanes with Rz (measured as the % of the full-length product) is indicated at the bottom.