Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast

Abstract

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.

Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae

“…Dangeard expressing different ELVd mutant forms indicates that the viroid sequence requirements for cleavage and ligation are different. More specifically, a quasi-double-stranded structure in the central part of the ELVd molecule (folded in the predicted minimum free-energy conformation) containing the ligation site in an internal loop motif seems involved in ELVd circularization in a chloroplastic context (33). This result suggested that an RNA ligase activity of chloroplastic localization, able to recognize the viroid ligation conformation, likely mediates ELVd circularization (33).…”

“…Reaction products were mixed with 1 volume of loading buffer (98% formamide, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.0025% xylene cyanol, 0.0025% bromophenol blue), denatured by heating for 2 min at 95°C, and separated by denaturing PAGE. ELVd (ϩ) RNAs were detected by Northern blot hybridization using a 32 P-labeled ELVd (Ϫ) RNA probe as previously described (33). In the circularization assay of the (ϩ) and (Ϫ) monomeric linear self-cleavage forms of all viroids (ASBVd, PLMVd, CChMVd, and ELVd), 1 mM ATP was replaced by 5 Ci of [␥-…”

“…More specifically, a quasi-double-stranded structure in the central part of the ELVd molecule (folded in the predicted minimum free-energy conformation) containing the ligation site in an internal loop motif seems involved in ELVd circularization in a chloroplastic context (33). This result suggested that an RNA ligase activity of chloroplastic localization, able to recognize the viroid ligation conformation, likely mediates ELVd circularization (33). A candidate for providing this activity is tRNA ligase, a conserved enzyme involved in maturation of nuclear tRNAs (1,13,31,46) that in plants is targeted to chloroplast, in addition to the nucleus and cytoplasm (14).…”

Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase

“…Reaction products were mixed with 1 vol of loading buffer [98% (vol/vol) formamide, 10 mM Tris·HCl (pH 8.0), 1 mM EDTA, 0.0025% xylene cyanol, 0.0025% bromophenol blue] and separated by denaturing PAGE in 5% (wt/vol) gels including 8 M urea in TBE buffer. RNAs were electroblotted to positively charged nylon membranes (Nytran SPC; Whatman), cross-linked by irradiation with 1.2 J/cm 2 UV light (Vilber Lourmat), and analyzed by hybridization using a complementary 32 Plabeled RNA probe as previously described (29). In some experiments, 1 mM ATP was excluded from the RNA circularization assay or replaced by 1 mM CTP, 1 mM GTP, 1 mM UTP, or all of the possible combinations of each two NTPs at 1 mM each.…”

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

“…Template switching also highlights the impact that replication can have on evolution because, in the three classes of small RNA replicons here compared, there are examples of mosaic sequences most likely emerging from recombination events between pre-existing RNAs; actually, the HDV genome itself has most stable secondary structure predicted for the viroid RNA. 79 Alternatively, circularization of chloroplastic viroids might be an RNA-mediated mechanism: (i) catalyzed by the hammerhead ribozyme and producing a 3',5'-phosphodiester linkage, thus resembling the mode of action of the hairpin ribozyme (see below) or (ii) autocatalyzed (self-ligation) and producing an atypical 2',5'-phosphodiester bond. 80 A critical assessment of these options has been presented elsewhere in reference 57.…”