Structural And Functional Study Of The Core Complex Of Chloroplast RNA Editosome

RNA editing is one of the post-transcriptional modification processes which alter the identity of nucleotides in RNA molecules by nucleotide insertion,deletion or replacement so that the information in the mature RNA differs from their genome encoded sequence.In chloroplasts and mitochondria of flowering plants,RNA editing alters C nucleotides to U nucleotides.To date,a series of RNA editing factors have been identified,including PPR?pentatricopeptide repeat?proteins,MORF?multiple organellar RNA-editing factors?proteins?also named RIP,RNA-editing factor interacting protein?.The PPR protein is a very large protein family in land plants.It can be further divided into P-type and PLS-type.Numerous studies have shown PLS-type PPR proteins are trans-factors that specifically recognize cis-elements of the target RNA.MORF is a newly identified protein family that are required for RNA editing.PLS-type PPR proteins and MORF proteins are expected to be the core complex of the hypothetical RNA editosome.Nevertheless,the actual function of MORF protein involved in RNA editing remains elusive,the structures of both MORF protein and PLS-type PPR protein are as yet unknown.Moreover,the molecular basis how MORF proteins cooperate with PLS-type PPRs remains unclear.To address this issue,we screened the interaction network between MORFs and PLS-type PPRs by yeast two hybrid and purified the recombinant MORF-PPR complex.Biochemical assay showed that MORF9 significantly increase the RNA-binding activity of LPA66,a member of the PLS-type PPR proteins.On this base,we determined the crystal structures of Arabidopsis chloroplast MORF9 and MORF2 by X-ray crystallography.To explore the molecular function of MORF proteins,we made numerous trials to crystallize the MORF-PPR complex,but failed due to the bad behavior of natural PLS-type PPR protein.Alternatively,we designed an artificial?PLS?₃PPR protein based on the consensus sequence of natural PLS-type PPR proteins,and then characterized the interactions between?PLS?₃PPR and MORF9.The RNA-binding activity of?PLS?₃PPR is drastically increased on MORF9 binding,which revealed a similar function of designer?PLS?₃PPR compared with that of natural PLS-type PPR.Finally,we successfully determined the crystal structure of?PLS?₃PPR–MORF9 complex.MORF9 induces significant compressed conformational changes of?PLS?₃PPR,revealing the molecular mechanisms by which MORF9-bound?PLS?₃PPR has increased RNA-binding activity.Our study not only identified the molecular mechanism of MORF protein in chloroplast RNA editing,but also deciphered the molecular architecture and working model of the core complex of RNA editosome.