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.

A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii

“…A striking feature of the predicted Mac1 polypeptide is the presence of two adjacent domains each with six or seven tetratricopeptide (TPR) repeats, most of which have features of the HAT subfamily (Half-A-TPR) (Supplemental Figure 1C). Members of this subfamily are implicated in RNA metabolism and some have been shown to associate with RNA in vivo or to bind RNA in vitro (Hammani et al, , 2014Loizeau et al, 2014). Indeed, we found the closest paralog of Mac1 in Chlamydomonas to be Mbb1, a TPR/HAT repeat protein involved in the stabilization or maturation of the psbB/T and psbH mRNAs (Vaistij et al, 2000a;Loizeau et al, 2014) (Supplemental Figure 3 and Supplemental Data Set 1).…”

“…Members of this subfamily are implicated in RNA metabolism and some have been shown to associate with RNA in vivo or to bind RNA in vitro (Hammani et al, , 2014Loizeau et al, 2014). Indeed, we found the closest paralog of Mac1 in Chlamydomonas to be Mbb1, a TPR/HAT repeat protein involved in the stabilization or maturation of the psbB/T and psbH mRNAs (Vaistij et al, 2000a;Loizeau et al, 2014) (Supplemental Figure 3 and Supplemental Data Set 1). The ortholog of Mbb1 in vascular plants, HCF107, is required for the stability of psbH transcripts (Felder et al, 2001;Sane et al, 2005;Hammani et al, 2012).…”

“…Mbb1, the closest paralog of Mac1 in Chlamydomonas, protects the psbB transcript by associating with its 59UTR (Vaistij et al, 2000b;Loizeau et al, 2014). This precedent and the presence of Mac1-dependent footprints matching the 59 end of psaC transcripts suggest that the target of Mac1 may be the 59UTR of psaC.…”

“…This mechanism can lead to the accumulation of RNA footprints, small RNA fragments that are protected by the respective RNA binding proteins (Pfalz et al, 2009;Ruwe and Schmitz-Linneweber, 2012;Zhelyazkova et al, 2012;Loizeau et al, 2014). Footprints that map to the 59end of the psaC transcripts could be identified in Chlamydomonas small RNA databases ( Figure 5; Supplemental Figure 5).…”

“…(A) sRNA sequence coverage graph of the psaC 59-region. Per-base read coverage within this region was extracted from sRNA sequencing databases (Ibrahim et al, 2010;Loizeau et al, 2014). Bars represent the psaC 59UTR and the coding sequence (the actual sequence is provided in Supplemental Figure 5).…”