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

Abstract

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b 6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687–697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Regulatory Sequences of Orthologous petD Chloroplast mRNAs are Highly Specific among Chlamydomonas Species

“…Mcd1 stabilizes the transcript by interacting directly or indirectly with the 5¢ terminal element I and blocking a 5¢-3¢ exoribonucleolytic activity (Drager et al, 1998(Drager et al, , 1999. Mcd1 specifically interacts with petD mRNA, as we showed with organellar microarrays (Erickson et al, 2005). The Mcd1 gene sequence was recently reported for C. reinhardtii and the predicted protein is novel (Murakami et al, 2005).…”

“…These data combined with those for DeG1 show that the C. eugametos petD terminal sequence is sufficiently different from C. reinhardtii that it does not function to stabilize mRNA in C. reinhardtii. One possible explanation for this difference is that the petD mRNA stability mechanisms are in general similar between these species, but the C. eugametos 1-57 sequence differences prevent it from interacting with C. reinhardtii stability factors, such as Mcd1 (Erickson et al, 2005;Murakami et al, 2005). A second possibility is that C. eugametos petD relies on an entirely different mechanism with non-homologous RNA cis elements and trans factors.…”

“…For example, the C. reinhardtii psbD and psbB chloroplast mRNAs parallel petD in having 5¢ terminal RNA stability elements and dependence on nucleus-encoded regulatory factors (Nac2 and Mbb1, respectively) that stabilize these transcripts against a 5¢-3¢ exoribonucleolytic degradation Vaistij et al, 2000a, b). However, the RNA stability sequences for these three mRNAs are different, and in mcd1 mutants psbD and psbB mRNAs accumulate normally (Erickson et al, 2005). The C. reinhardtii chloroplast psbC mRNA also has a 5¢ terminal stability sequence (Zerges et al, 2003).…”

“…Often, these elements function by interacting with nucleus-encoded trans-acting factors, and many work through gene-specific processes (Barkan and Goldschmidt-Clermont, 2000;Erickson et al, 2005;Murakami et al, 2005;Zerges, 2000).…”

Processing, degradation, and polyadenylation of chloroplast transcripts

“…Known targets include petA, psbB-psbT, petD, psbC, atpB, and psbD (Barkan and Goldschmidt-Clermont 2000). The specificity of such mutants is somewhat presumptive, since in only one case was each chloroplast transcript checked in the mutant background; a microarray analysis of the petD mutant mcd1 confirmed its specificity (Erickson et al 2005).…”