Evidence for multiple RNA-degrading pathways in the Chlamydomonas chloroplast

Maria L. Salvador1, Loreto Suay1, and Uwe Klein2

1) Department of Biochemistry and Molecular Biology, University of Valencia, 46100 Valencia, Spain 2) Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway

The goal of the present study was to learn more about the nucleolytic pathway(s) by which transcripts are degraded in the chloroplast of Chlamydomonas reinhardtii. To this end the 5' untranslated region (5' UTR) of the rbcL gene, fused to the uidA (GUS) reporter gene, was modified in various ways and the effect of the modifications on levels of GUS transcripts in the dark determined. First, single-stranded tails of up to 19 extra nucleotides were added to the 5' terminus of the rbcL 5' UTR in order to destabilize the reporter gene transcripts. Tails longer than 8 nucleotides were found to completely destabilize the transcripts suggesting that degradation is initiated somewhere along the extra nucleotide sequence. Placing a poly(G) tract -- known to block exoribonucleolytic degradation -- at the 5' terminus of destabilized transcripts resulted in very stable transcripts that accumulated to even higher levels than unmodified control transcripts. This suggests a role of a transcript's 5' terminus in the initial nucleolytic attack. Poly(G) tracts inserted in various positions downstream of the 5' terminus also blocked degradation of destabilized transcripts. In all these cases the 5' ends of the transcripts corresponded to the 5' ends of the poly(G) tracts, as shown by primer extension analysis, confirming earlier reports of the involvement of a 5' -> 3' exoribonuclease in the normal RNA degradation pathway. However, not all transcripts that were destabilized by changes in the rbcL 5' UTR sequence, could be rescued by inserting poly(G) tracts but were completely degraded. Furthermore, degradation of poly(G)-rescued transcripts could still be enhanced by illuminating the cells implying a light-triggered component of RNA degradation that cannot be blocked by poly(G) tracts. These results strongly suggest multiple pathways for degradation of transcripts in the chloroplast of Chlamydomonas reinhardtii.

e-mail address of presenting author: uwe.klein@imbv.uio.no