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cpRNPs as regulators of chloroplast gene expression

The cpRNPs are proteins unique to chloroplasts and consist of two consensus sequence-type RNA recognition motifs (RRM) and an acidic N-terminal domain. They have a strong affinity to RNA homopolymers, poly (G) and poly (U), rather than single-stranded (ss) DNA and double-stranded (ds) DNA in vitro (reviewed in Nakamura et al., 2004). Therefore, they are thought to play a role in RNA metabolism in chloroplasts. Further in vitro studies reported that cpRNPs are required for 3’-end formation of several mRNAs (Schuster and Gruissem, 1991), for RNA editing (Hirose and Sugiura, 2001), and for RNA stability (Nakamura et al., 2001). What distinguishes cpRNPs from PPRs and most other plastid-localized RNA binding proteins is that these proteins are extremely abundant, outnumbering all plastid mRNAs combined and that they are heavily regulated transcriptionally and post-translationally (Nakamura et al., 2001; Kleffmann et al., 2007). For example, while most PPR family members are constitutively expressed, cpRNPs accumulate in a light-dependent, rhythmic fashion (Raab et al., 2006). Light also induces cpRNP phosphorylation and thus modulates their affinity for RNA (Lisitsky and Schuster, 1995). These studies suggest that cpRNPs could be factors connecting light signals with responses on the RNA level thereby ultimately impacting the light-dependent expression of the photosynthetic apparatus. Such a connection has been known for a long time (e.g. Deng et al., 1989), but the underlying factors are almost entirely unknown.

This project asks: What RNA populations are defined by cpRNPs and what functions do cpRNPs have in vivo? What role does cpRNP phosphorylation play for light-dependent gene expression in chloroplasts?

We have so far isolated T-DNA insertion mutants for nine of the ten known Arabidopsis cpRNPs. In mutants of CP31A, we analyzed the status of all known chloroplast RNA editing sites. We identified RNA editing defects in 13 of the 34 sites analysed (Tillich et al., 2009). Also, we found a surprisingly radical loss of the ndhF RNA, but not of other mRNAs. ndhF encodes subunit of NDH dehydrogenase complex that is implicated in cyclic electrone transport and stress-related responses of the thylakoid membrane electrone transport chain. To determine RNA targets of cpRNPs, we carried out RIP-Chip experiments with polyclonal antibodies. These experiments indicate that cpRNPs associate with all chloroplast mRNAs, but are not attached to tRNAs or rRNAs (Kupsch et al., 2012). This apparent non-specific binding to most chloroplast RNAs is contrasting the specificity of RNA processing defects observed in cpRNP mutants and cannot be explained at the moment. We speculate that cpRNPs operate similar to their nuclear relatives, the ubiquitous heterogenous nuclear ribonucleoproteins (hnRNPs). These latter proteins act in concert in a combinatorial fashion to determine which RNA processing event takes place on which specific message and we hypothesize that cpRNPs act in a similar way.

Recently, outside of the EN project, we have shown that cpRNPs are important for cold stress tolerance in Arabidopsis. Further stress tests of cpRNP mutants are currently under way.