Abstract
Chloroplast genes are transcribed as polycistronic precursor RNAs that give rise to a multitude of processing products down to monocistronic forms. Translation of these mRNAs is realized by bacterial type 70S ribosomes. A larger fraction of these ribosomes is attached to chloroplast membranes. This study analyzed transcriptome-wide distribution of plastid mRNAs between soluble and membrane fractions of purified plastids using microarray analyses and validating RNA gel blot hybridizations. To determine the impact of light on mRNA localization, we used etioplasts, greening plastids and mature chloroplasts from Zea mays as a source for membrane and soluble extracts. The results show that the three plastid types display an almost identical distribution of RNAs between the two organellar fractions, which is confirmed by quantitative RNA gel blot analyses. Furthermore, they reveal that different RNAs processed from polycistronic precursors show transcript-autonomous distribution between stroma and membrane fractions. Disruption of ribosomes leads to release of mRNAs from membranes, demonstrating that attachment is likely a direct consequence of translation. We conclude that plastid mRNA distribution is a stable feature of different plastid types, setting up rapid chloroplast translation in any plastid type.
Highlights
Subcellular RNA localization is an important means of gene regulation in eukaryotic organisms [1], and in bacteria [2]
A large fraction of all Chlamydomonas chloroplast 70S ribosomes is attached to thylakoid membranes, as evidenced both by electron microscopy as well as cell fractionation [7,8,9]
We prepared membranes and stroma extracts from isolated plastids from 9-day-old Zea mays seedlings that were either grown in the dark, from greening seedlings, or from plants grown under standard long-day conditions
Summary
Subcellular RNA localization is an important means of gene regulation in eukaryotic organisms [1], and in bacteria [2]. A large fraction of all Chlamydomonas chloroplast 70S ribosomes is attached to thylakoid membranes, as evidenced both by electron microscopy as well as cell fractionation [7,8,9] This suggested early on that chloroplast mRNAs are translated in close association with chloroplast membranes. Since many chloroplast mRNAs encode integral membrane proteins, it was assumed that translating ribosomes get trapped on membranes because the nascent protein chain is translated directly into the thylakoid membrane This is supported by the finding that membrane attachment of RNAs depends on active translation [10] and is proportional to overall protein synthesis within thylakoid membranes [11]. In rare cases such associations are subject to developmental change, but in general, membrane-enrichment of plastid RNAs is constant during chloroplast development
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