Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts

Abstract

Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.psbA mRNA-associated proteome 371 d Cutoff: >3-fold enrichment in the designer SCD11 pull-down data (McDermott et al., 2019). Proteins not found in the chloroplast were excluded. *Not detected in control (pre-immune serum for HCF173 co-immunoprecipitates, leaf proteome for PIRP, anti-FLAG pull-down of Col-0 for dPPR).

The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA

“…Consistently, a number of proteins have been co-purified with the psbA mRNA in Chlamydomonas, spinach, Arabidopsis and maize, and or have been identified by genetic analyses [27][28][29][30][31][32][33][34][35]. Among the proteins co-precipitating with the psbA mRNA was CP33B (AT2G35410) [9]. In contrast to other cpRNPs, CP33B appears to have a clear preference for the psbA mRNA and does show comparatively little binding to other mRNAs [9].…”

“…Among the proteins co-precipitating with the psbA mRNA was CP33B (AT2G35410) [9]. In contrast to other cpRNPs, CP33B appears to have a clear preference for the psbA mRNA and does show comparatively little binding to other mRNAs [9]. We here analyzed the binding preference of CP33B in more detail and identified a main target sequence motif in vitro.…”

“…We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B's preference for psbA, suggesting that there are other determinants of specificity in vivo.Plants 2020, 9, 367 2 of 15 of target mRNAs for various cpRNPs [7][8][9]. rRNAs and intron-less tRNAs are not or only weakly bound [6,7,10].…”

“…Plants 2020, 9, 367 2 of 15 of target mRNAs for various cpRNPs [7][8][9]. rRNAs and intron-less tRNAs are not or only weakly bound [6,7,10].…”

The chloroplast ribonucleoprotein CP33B quantitatively binds thepsbAmRNA

“…Consistently, a number of proteins have been co-purified with the psbA mRNA in Chlamydomonas, spinach, Arabidopsis and maize, and or have been identified by genetic analyses (Danon and Mayfield, 1991;Alexander et al, 1998;Shen et al, 2001;Ossenbuhl et al, 2002;Schult et al, 2007;Link et al, 2012;Bohne et al, 2013;McDermott et al, 2019;Williams-Carrier et al, 2019). Among the proteins co-precipitating with the psbA mRNA was CP33B (AT2G35410; Watkins et al, 2019). In contrast to other cpRNPs, CP33B appears to have a clear preference for the psbA mRNA and does show comparatively little binding to other mRNAs .…”