Chloroplast Ribonucleoproteins Function as a Stabilizing Factor of Ribosome-free mRNAs in the Stroma

Abstract

Post-transcriptional RNA processing is an important step in the regulation of chloroplast gene expression, and a number of chloroplast ribonucleoproteins (cpRNPs) are likely to be involved in this process. The major tobacco cpRNPs are composed of five species: cp28, cp29A, cp29B, cp31, and cp33 and these are divided into three groups (I, II, and III). By immunoprecipitation, gel filtration, and Western blot analysis, we demonstrated that these cpRNPs are abundant stromal proteins that exist as complexes with ribosome-free mRNAs. Many ribosome-free psbA mRNAs coprecipitate with cpRNPs, indicating that the majority of stromal psbA mRNAs are associated with cpRNPs. In addition, an in vitro mRNA degradation assay indicated that exogenous psbA mRNA is more rapidly degraded in cpRNP-depleted extracts than in nondepleted extracts. When the depleted extract was reconstituted with recombinant cpRNPs, the psbA mRNA in the extract was protected from degradation to a similar extent as the psbA mRNA in the nondepleted extract. Moreover, restoration of the stabilizing activity varied following addition of individual group-specific cpRNPs alone or in combination. When the five cpRNPs were supplemented in the depleted extract, full activity was restored. We propose that these cpRNPs act as stabilizing factors for nonribosome-bound mRNAs in the stroma.