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Nucleic Acids Res. 2014 Mar;42(5):3286-97. doi: 10.1093/nar/gkt1272. Epub 2013 Dec 11.

Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas.

Author information

1
Department of Botany and Plant Biology and Institute of Genetics and Genomics in Geneva University of Geneva, CH-1211 Geneva 4, Switzerland and Institute of Biology, Molecular Genetics, Humboldt University of Berlin, D-10115 Berlin, Germany.

Abstract

Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5'-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5' UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5' UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5' UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

PMID:
24335082
PMCID:
PMC3950674
DOI:
10.1093/nar/gkt1272
[Indexed for MEDLINE]
Free PMC Article

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