A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii

Abstract

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Microarray Analysis Confirms the Specificity of a Chlamydomonas reinhardtii Chloroplast RNA Stability Mutant

“…This strain is a double mutant, carrying both the mcd1-2 petD instability mutation and the mcd2-1 mutation that suppresses mcd1-2 and restores photosynthesis (Esposito et al, 2001). In the double mutant approximately 10% of wild-type levels of petD mRNA and approximately 50% of SUIV accumulate.…”

“…This 5#-3# degradation may also function to process petD pre-mRNA and form the mature 5# end. The RNA instability phenotype of mcd1-2, but not mcd1-1, can be partially suppressed by a mutation in the unlinked Mcd2 gene (mcd2-1; Esposito et al, 2001).…”

Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii

“…Furthermore, numerous photosynthetic (PS1) suppressors have been recovered from screening of PS2 strains (e.g. Girard-Bascou et al, 1992;Levy et al, 1997;Bernd and Kohorn, 1998;Nickelsen, 2000;Esposito et al, 2001;Li et al, 2002).Recovery of wild-type alleles of genes mutated in PS2 strains has been successful, since both genomic complementation with selection on minimal medium or identification of DNA flanking an insertional mutant site are relatively straightforward although perhaps tedious methods (e.g. Gumpel et al, 1995;Boudreau et al, 2000;Vaistij et al, 2000;Auchincloss et al, 2002;Dauvillee et al, 2003).…”

“…However, in cases where PS1 suppressors have been recovered from PS2 strains or where a trait otherwise cannot be selected on minimal medium, recovery of the mutation requires other methods. In the case of PS1 suppressors, for example, a recessive suppressor would yield a PS2 phenotype upon complementation, and even dominant suppressors such as mcd2, which suppresses the nuclear mcd1-2 mutation responsible for instability of the chloroplast petD mRNA (Esposito et al, 2001), require construction of a genomic library from the suppressed strain if they are to be cloned by complementation. Another example is the xanthophyll cycle mutant npq1, which is defective in nonphotochemical quenching (Niyogi et al, 1997).…”

“…Furthermore, numerous photosynthetic (PS1) suppressors have been recovered from screening of PS2 strains (e.g. Girard-Bascou et al, 1992;Levy et al, 1997;Bernd and Kohorn, 1998;Nickelsen, 2000;Esposito et al, 2001;Li et al, 2002).…”