Spontaneous Dominant Mutations in Chlamydomonas Highlight Ongoing Evolution by Gene Diversification

Abstract

We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins.

FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain

“…This resemblance between RAP domains and these nucleases is apparently highly conserved, as RAP domains in plant chloroplast proteins also resemble PD-(D/E)-XK nucleases, specifically including the VSR endonucleases (20). …”

Engineering RNA‐binding proteins with diverse activities

“…Although initially identified as DNA‐binding transcription terminators, recent studies have shown that some mTERF proteins can bind RNA to mediate group II intron splicing in mitochondria and chloroplasts or mediate mitochondrial ribosomal RNA (rRNA) methylation . Another example is octotricopeptide repeat (OPR) protein that can regulate the mRNA processing or rRNA maturation in chloroplasts . Like PUF and PPR, these helical repeat proteins probably also bind RNA in a modular fashion, and thus may be used as programmable RNA‐binding scaffold in artificial proteins in the future.…”

“…43 Another example is octotricopeptide repeat (OPR) protein that can regulate the mRNA processing or rRNA maturation in chloroplasts. [44][45][46] Like PUF and PPR, these helical repeat proteins probably also bind RNA in a modular fashion, and thus may be used as programmable RNA-binding scaffold in artificial proteins in the future.…”

Twice as NCC: Two Octotricopeptide Repeat Proteins and the Regulation of Chloroplast Gene Expression

“…The dominant, nuclear mutation ncc2 results in loss of petA mRNA, but not psbB. (Reprinted from Boulouis et al [2015], Figure 2E. …”

“…To screen for mutants in nuclear genes that affect chloroplast gene expression in Chlamydomonas reinhardtii, Boulouis et al (2015) expressed psbB, which encodes a core component of photosystem II, under the control of the 5# untranslated region (UTR) of the chloroplast gene petA, which encodes a subunit of cytochrome b 6 f. Expression from the 5#petA-psbB construct does not produce enough photosystem protein for the cells to grow photoautotrophically. The authors identified a spontaneous, dominant mutant that allowed photoautotrophic growth, which they named ncc2 (nuclear control of chloroplast gene expression2).…”