Can the import of mRNA into chloroplasts be mediated by a secondary structure of a small non-coding RNA?

Abstract

The import of diverse nucleus-encoded proteins into chloroplasts is crucial for plant life. Although this crosstalk is mainly dependent on specific transit peptides, it has been recently reported that a non protein-coding RNA (ncRNA) based on a viroid-derived sequence (vdRNA) and acting as a 5´UTR-end mediates the functional import of GFP-mRNA into chloroplasts. This observation unearths a novel plant cell signaling pathway able to control the accumulation of the nuclear-encoded proteins in this organelle. The mechanisms regulating this chloroplast-specific localization remain yet unclear. To unravel the functional nature of this chloroplastic signal, here we dissect the 5´UTR-end responsible for the chloroplast targeting. A confocal microscopy analysis in Nicotiana benthamiana leaves of the transcripts expression carrying partial deletions of the 5`UTR-end indicate that an internal 110 nucleotides-length fragment is sufficient to mediate the traffic of functional GFP-mRNA into chloroplasts. However, the capability of this motif to act as a chloroplastic localization signal was enhanced when fused to either the 5` or the 3`region of the vd-5´UTR sequence. These findings suggest that the chloroplast-specific RNA targeting is dependent on a structural motif rather than on the RNA sequence.

Figure 1

Physical map of the partially deleted vd-5′UTR/GFP constructs used in this work. The full vd-5′UTR chloroplastic signal (AN-HM136583) was dissected into three arbitrary regions: I (108 nt) left, II (110 nt) internal and III (112 nt) right. Those constructs containing different combinations of these partial vd-5′ UTR ends transcriptionally fused to the GFP-cDNA are shown in detail.

Figure 2

Region 2 of the vd-5′UTR end mediates chloroplast-specific trafficking. Observation at confocal microscope of the N. benthamiana leaves expressing the different combinations of the deleted vd-5′UTR end transcriptionally fused to the GFP (see the text for details). The unmodified GFP and the full vd-5′UTR /GFP constructs were used as cellular localization controls.