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

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.

Figure 1.

sRNAs correspond to the 5'-ends of chloroplast mRNAs in Chlamydomonas. (A) The sequences corresponding to four chloroplast sRNAs (Supplementary Table S1) are highlighted in bold. The position of the 5'-ends as mapped by RACE is shown with arrowheads. The numbers above the arrowheads indicate the number of 5'-RACE clones that end at the respective sites. Most clone ends coincide with sRNA 5'-ends. (B) Sequence alignment of the psbB 5' and psbH 5' sRNAs. Asterisks indicate conserved positions. (C) Autoradiograms of an RNA blot of chloroplast RNA from the WT and the mbb1 mutant hybridized with the radioactive probes indicated above the panels. The arrows indicate the positions of the sRNAs. Low molecular weight RNA was enriched from total RNA and separated on a 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis containing 8 M urea. A fluorescence image of the gel lanes stained with ethidium bromide is shown as loading control in the leftmost panel. The probes were single-stranded DNA oligonucleotides (≤25 nt) antisense to the sRNAs. The same membrane was repeatedly probed and stripped as described in Materials and Methods section.

Figure 2.

Mutational analysis of the psbB 5' UTR. (A) Map of the psbB/psbT/psbH locus. The gray bars represent the open reading frames of psbB, psbT and psbH, and below the line of the putative psbN, which would be transcribed from the opposite strand. Restriction sites relevant to this work are indicated at the top, and the probes used for RNA blot hybridization are shown below as white bars. (B) The 5' UTRs of the psbB transcripts. The major form of the psbB 5' UTR begins at −35 relative to the start codon. A minor form, which is two orders of magnitude less abundant, begins at −147 (19). (C) Linker-scan mutagenesis of the psbB 5' UTR. The sequence of the WT is shown at the top with the translation start codon of psbB highlighted in bold and the S-box underlined. The corresponding sRNA (psbB 5') is shown in lowercase above the sequence. The black arrow shows the positions of the major 5'-end at −35. The sequences of the chloroplast transformation vector p38ANco and of the eight linker-scan mutants are aligned below, with the ApaI sites in italics and with dots representing unchanged bases.

Figure 3.

RNA analysis of the psbB 5' UTR mutants. (A) RNA blot hybridization analysis of the psbB/T/H transcripts. Total RNA was analyzed by agarose gel electrophoresis and blot hybridization using the probes indicated to the left of each panel. A probe for exon 3 of psaA was used as a control. (B) Primer extension analysis of the psbB RNAs. Total RNA was used as a template for reverse transcription using oligonucleotide primers specific for psbB, psbD or both as indicated at the top of the panel. The psbD primer serves as an internal control. Owing to the abundance of psbD mRNA, background precludes the detection of the longer form of psbB RNA starting at −147, which is two orders of magnitude less abundant than the form at −35 (19). The panel on the right shows sequencing reactions of the psbB 5' UTR with the psbB primer.

Figure 4.

Mutational analysis of the conserved S-box in the psbH 5' UTR. (A) Mutagenesis of the psbH 5' UTR. The sequence of the psbB 5' UTR is shown at the top for comparison, and the sequence of the psbH 5' UTR below it, with the translation start codons in bold. The conserved ‘S-box’ is highlighted in bold and underlined. The sequences of the sRNAs are shown in lowercase above the respective sequences. The black arrows show the positions of the major 5'-ends (see Figure 2 and Supplementary Figure S3). The transformation vector (p41A) carries a selectable aadA spectinomycin resistance cassette inserted downstream of psbH (Materials and Methods). The Chlamydomonas host strain (ΔH) carries a substitution of the psbH gene and its 5' UTR to ensure that transformants carrying the aadA marker also bear the desired mutation. (B) Immunoblot analysis of PSII components. Total protein extracts were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with the antibodies indicated on the left. From left to right, the samples are the WT, the mbb1-222E mutant (mbb1), the ΔH transformation host (ΔH), the psbB m26-31 mutant (m26-31), the transformation host rescued with a WT psbH vector (p41A) and the three mutants shown in (A). (C) RNA blot hybridization analysis of the psbB/T/H transcripts. Total RNA was analyzed by agarose gel electrophoresis and RNA blot hybridization using the psbH and psbB probes as indicated on the left. A probe for psaA was used as a control. (D) Mapping of the psbH transcripts. The schematic map shows the positions of the 5'-end at −54/−53 with a black arrow and of the 3' termini with white arrows [according to (30)]. The upstream probe (5') and coding sequence probe (cod) are depicted below the map as open bars. Total RNA from the WT and a representative mutant (ApaS) was analyzed by agarose gel electrophoresis and RNA blot hybridization using the probes indicated at the bottom of each panel.

Figure 5.

Electrophoretic mobility shift assay with Mbb1-HA immunoprecipitates. (A) Binding of Mbb1-HA to the 5' UTR of psbB. A radiolabeled probe corresponding to the mature psbB 5' UTR was incubated with increasing amounts of Mbb1-HA immunoprecipitate (0.1, 1, 2.5 and 4.5 µg) and resolved by native polyacrylamide gel electrophoresis. Bound (B) and unbound (U) probes are marked. A similar experiment was performed with increasing amounts of Sbp-HA (sedoheptulose 1,7 bisphophatase) as a negative control. (B) Competition of RNA binding with WT or mutant psbB 5' UTR. One microgram of Mbb1-HA protein extract was incubated with a radiolabeled probe corresponding to the WT psbB 5' UTR and an excess (10-, 50- or 100-fold) of an unlabeled competitor corresponding either to the m26-31 psbB 5' UTR (left) or the WT psbB 5' UTR (right). (C) Binding of Mbb1-HA to the 5' UTR of psbH. A radiolabeled probe corresponding to the mature psbH 5' UTR was incubated with increasing amounts of Mbb1-HA immunoprecipitate as in (A). (D) Competition of RNA binding with WT or mutant psbH 5' UTR. Competition was performed as in (B) with WT or mutant (ApaS) psbH 5' UTR unlabeled RNA.