A viroid-derived system to produce large amounts of recombinant RNA in Escherichia coli

Abstract

Viruses have been engineered into useful biotechnological tools for gene therapy or to induce the synthesis of products of interest, such as therapeutic proteins and vaccines, in animal and fungal cells, bacteria or plants. Viroids are a particular class of infectious agents of higher plants that exclusively consist of a small non-protein-coding circular RNA molecule. In the same way as viruses have been transformed into useful biotechnological devices, can viroids be converted into beneficial tools? We show herein that, by expressing Eggplant latent viroid (ELVd) derived RNAs in Escherichia coli together with the eggplant tRNA ligase, this being the enzyme involved in viroid circularization in the infected plant, RNAs of interest like aptamers, extended hairpins, or other structured RNAs are produced in amounts of tens of milligrams per liter of culture. Although ELVd fails to replicate in E. coli, ELVd precursors self-cleave through the embedded hammerhead ribozymes and the resulting monomers are, in part, circularized by the co-expressed enzyme. The mature viroid forms and the protein likely form a ribonucleoprotein complex that transitorily accumulates in E. coli cells at extraordinarily amounts.

Figure 1

ELVd (sequence variant AJ536613) folded in the predicted secondary structure of minimum free energy. The domain and self-cleavage site of the hammerhead ribozyme are indicated on yellow background and by a red arrowhead, respectively. The internucleotide position U245-U246 where the RNAs of interest were grafted is indicated by a blue arrow.

Figure 2

Analysis of RNAs accumulated in E. coli cells that co-express ELVd RNA and eggplant tRNA ligase. RNAs were separated by PAGE, stained first with ethidium bromide (a,c and e), and transferred next to membranes, hybridized with a radioactive probe complementary to ELVd RNA and autoradiographed (b and d). (a and b) RNAs separated by single denaturing PAGE. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 to 4, RNAs from the E. coli cells transformed with pLELVd (lane 2), pLELVd and p15tRnlSm (lane 3) and p15tRnlSm (lane 4). (c and d) RNAs from the E. coli co-transformed with pLELVd and p15tRnlSm separated by two-dimension denaturing PAGE in two different buffer conditions, as indicated. (e) RNAs separated by single denaturing PAGE. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 to 10, RNAs from independent E. coli clones transformed with pLELVd (lanes 2 to 4), pLELVd and p15tRnlSm (lanes 5 to 7), and pLELVd and p15mCherry (lanes 8 to 10). In the different panels, the positions of the circular and linear ELVd RNAs, and of E. coli 23S and 16S rRNAs, are indicated. (a–d) E. coli cultures were grown at 25 °C and induced with 0.4 mM IPTG at OD₆₀₀ 0.6. Bacteria were harvested at 10 h post-induction. (e) E. coli cultures were grown at 37 °C and induced with 0.1 mM IPTG at OD₆₀₀ 0.1. Bacteria were harvested at 8 h post-induction. Each lane contains an aliquot of RNA that corresponds to 0.8 ml of culture in all cases.

Figure 3

Polarity analysis of the ELVd RNAs that accumulated in E. coli cells that co-express ELVd RNA and eggplant tRNA ligase. RNAs were separated by PAGE, stained with ethidium bromide, transferred to membranes and hybridized with RNA radioactive probes to detect ELVd RNAs of plus (a) and minus (b) polarities. Lane 1, total RNAs purified from an ELVd-infected eggplant; lanes 2 to 5, ELVd RNAs purified from E. coli cells transformed with pLELVd and p15tRnlSm. To better appreciate the differential hybridization with both ELVd probes, ELVd RNAs from E. coli (lane 5) were loaded serially diluted 1/10 in lane 4, 1/100 in lane 3 and 1/1000 in lane 2. The positions and sizes (in nt) of RNA markers are indicated on the left of both panels. The positions of monomeric circular and linear ELVd RNAs are indicated on the right of both panels.

Figure 4

Recombinant ELVd-Spinach RNA production in E. coli using the viroid-derived system. (a) Time-course analysis of the RNA that accumulated in E. coli cells that co-expressed ELVd-Spinach RNA and eggplant tRNA ligase. Aliquots were taken from the culture at different time points and RNA was extracted. RNAs were separated by denaturing PAGE and the gel was stained with ethidium bromide. Lanes 1 to 8, RNAs from the aliquots taken at 0, 2, 4, 6, 8, 10, 12 and 24 h post-induction of tRNA ligase expression. The positions of the circular and linear ELVd-Spinach RNAs are indicated on the right. E. coli cultures were grown at 37 °C in TB medium and induced with 0.1 mM IPTG at OD₆₀₀ 0.1 mM. Each lane contains the RNA that corresponds to 0.4 ml of culture. (b) Pictures of the sedimented E. coli cells that co-expressed tRNA ligase and an empty vector, ELVd or ELVd-Spinach, as indicated (left), and the corresponding RNA extracts (right). The lower pictures were taken under UV illumination using a GFP filter. DFHBI was added to either the E. coli culture before cell recovery (left) or the RNA extract (right).

Figure 5

Production of extended hairpins and a crRNA in E. coli using the viroid-derived system. RNAs were extracted from the aliquots of E. coli cultures taken at 15.5 h post-inoculation, separated by denaturing PAGE, and gels were stained with ethidium bromide. (a) Hairpin RNAs were produced in the RNase III-deficient E. coli strain HT115(DE3) co-transformed with p15LtRnlSm and the different pLELVd-hairpin. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 to 6, RNAs from the E. coli transformed to express empty ELVd (lane 2) and the different ELVd forms that included hairpin RNAs of 40, 60, 80 and 100 nt, as indicated (lanes 3 to 6). The positions of the circular empty ELVd and the different chimeric ELVd-hairpin RNAs are indicated on the right. E. coli cultures were grown at 37 °C in TB medium. Each lane contains an aliquot of RNA that corresponds to 0.4 ml of culture. (b) crRNA was produced in E. coli BL21(DE3) co-transformed with p15LtRnlSm and pLELVd-crRNA. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 and 3, RNAs from the E. coli transformed to express empty ELVd and the chimeric ELVd-crRNA, respectively. The positions of the circular empty ELVd and the chimeric ELVd-crRNA are indicated on the right. E. coli cultures were grown at 37 °C in TB medium and cells harvested at 15.5 h post-inoculation. Each lane contains an aliquot of RNA that corresponds to 0.8 ml of culture.