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

Karen Loizeau 1 y Yujiao Qu 0 y Se bastien Depp 1 Vincent Fiechter 1 Hannes Ruwe 0 Linnka Lefebvre-Legendre 1 Christian Schmitz-Linneweber 0 Michel Goldschmidt-Clermont 1 0 Institute of Biology, Molecular Genetics, Humboldt University of Berlin , D-10115 Berlin, Germany 1 Department of Botany and Plant Biology and Institute of Genetics and Genomics in Geneva University of Geneva , CH-1211 Geneva 4, Switzerland - 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 50-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 50 UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 50 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 cisacting element at the extremity of the psbH and psbB 50 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. INTRODUCTION In the chloroplast, post-transcriptional steps play a major role in the control of gene expression. Many chloroplast genes are part of polycistronic transcription units, and RNA maturation is complex. It involves endonucleolytic and exonucleolytic processing at the 50-end, the 30-end and in intergenic spacers, intron splicing and in plants, RNA editing (14). These maturation events in turn influence messenger RNA (mRNA) translation (57). RNA maturation, RNA stability and translation are regulated by developmental programs and by environmental factors such as light or nutrient availability (6,8,9). Numerous nucleusencoded factors are imported in the chloroplast where they govern these post-transcriptional events (4,10). Most of these factors are highly specific and generally target only one or a few genes. A prominent example for such RNA-binding proteins is the members of the helicalrepeat protein super-family, which fulfill various tasks for the maturation of organellar RNAs and include pentatricopeptide repeat (PPR), octotricopeptide repeat (OPR) or TPR/HAT (tetratricopeptide repeat/half a tetratricopeptide repeat) proteins (1116). The prototypical example of helical-repeat proteins is Pumilio, where each repeat is composed of three alpha-helices that interact to provide a super-helical scaffold. Each repeat presents specific amino acid residues that bind to 1 nt of the RNA substrate (17). The OPR family has expanded during the evolution of Chlamydomonas reinhardtii, whereas it is the PPR family that is most prominent in the higher-plant lineages (1214). Members of these helical-repeat protein families can protect chloroplast RNAs against exonucleolytic degradation by tightly yThese authors contributed equally to the paper as first authors. binding sequences in the UTRs and thus increase the stability of their substrates (1824). A well-studied example is the binding of PPR10 to its target sequences, which impedes the progression of both 50- and 30-exonucleases and can thus protect either the downstream or the upstream RNA, respectively (22,23). Intriguingly, PPR10 as well as other helical repeat proteins generate short, 15 30-nt-long sRNAs simply by protecting the bound RNA segment, i.e. the footprint, against exonucleolytic degradation (2224). Almost 100 such sRNAs are found in plant chloroplast transcriptomes, many corresponding to ends of transcripts and to known or presumed protein binding sites (2527). Consequently, sRNAs can be used as a proxy to identify binding sites of RNA-binding proteins. To date, sRNA data sets have been presented for different species of angiosperms, namely, barley, maize, Arabidopsis and Chinese cabbage (25,26,28). Whether sRNAs are present in chloroplasts of other lineages is at present unclear, although this is suggested by the wide evolutionary distribution of chloroplast-targeted helical-repeat proteins. Here, we identify chloroplast sRNAs of the green alga C. reinhardtii in public data sets from high-throughput RNA-sequencing experiments. We show that some sRNAs co-localize with transcript ends and can be detected by RNA gel blot analysis. To investigate their biological significance, we focus on two sRNAs that map to the psbB/psbT/psbH gene cluster, which is transcribed as a unit and processed to give rise to the monocistronic psbB and dicistronic psbB/T mRNAs, as well as to several forms of psbH RNA, all of which encode subunits of PSII (29,30). The nucleus-encoded factor Mbb1 is specifically required for the stable accumulation of all the transcripts from this cluster (29,31). The analysis of reporter constructs has shown that Mbb1 acts through the 50 untranslated region (50 UTR) of psbB, and there is genetic evidence that it also acts directly on psbH (19). Mbb1 is one of the rare RNA-binding proteins in chloroplasts for which an ortholog can be identified in higher plants. This ortholog, named HCF107 (high chlorophyll fluorescence 107), is required for expression of psbB and psbH (32,33). In the hcf107 mutant, RNA processing upstream of psbH is deficient and its translation is impaired. Translation of psbB is also defective in this mutant (32), even though the pattern of psbB transcripts appears normal. In vitro assays have demonstrated that recombinant HCF107 binds the 50-end of the psbH transcript and can protect it against exonucleolytic degradation from either the 50 or the 30 side (24). An sRNA representing the footprint of HCF107 is detected in vivo. Thus, the TPR/HAT protein HCF107 seems to act similarly to PPR proteins like PPR10, in line with their similar predicted helical-repeat structure (34). Here we show that the sRNAs mapping to the ends of the 50 UTRs of psbH and psbB are missing in the mbb1 mutant, suggestive of a direct functional link between Mbb1 and these short RNA segments. Using chloroplast site-directed mutagenesis, we demonstrate the importance of the corresponding sequence elements for mRNA stability in vivo by a systematic genetic survey of the entire psbB 50 UTR and of conserved sequences in the psbH 50 UTR. Association of Mbb1 with this RNA sequence element is demonstrated by in vitro binding assays. MATERIALS AND METHODS Strains and media The C. reinhardtii mbb1-222E mutant st (...truncated)