The L motifs of two moss pentatricopeptide repeat proteins are involved in RNA editing but predominantly not in RNA recognition

Abstract

Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

“…Even more so, the unexpected non-canonical preference of PPR P-6ND for guanosine instead of the expected uridine in PPR56 appears to be even enhanced by mutation of the upstream PPR S-7TD > TN (Figure 8 ). Notable additional elements evidently contributing to positional nucleotide preferences in the target RNA are selectivity also influenced by L-type PPRs ( 28 , 59 ) and the evident preferences in positions -3 to -1 upstream of editing sites as documented in our off-target studies and likely due to the specific makeup of the E and DYW domains, in full accord with earlier reports ( 53–55 ).…”

“…However, with this low number of detected off-targets, PPR65 yielded much less than PPR56 and, with the above caveat on different sorting approaches and limited editing observed for PPR65 in the tested setup, this would be in accordance with the surprisingly few off-targets previously observed in E. coli ( 28 ). On the one hand, this different behavior of the two editing factors may well be caused by differential and stronger sequence selectivity exerted not only by the P- and S-type PPRs alone, but also by the respective L-type PPRs and the E1/E2 and DYW domains, which are meantime known to contribute to target selection in a presently not understood way ( 53–55 , 59 ). However, taking into account that we also observed dramatically reduced or significantly increased overall numbers of off-targets upon the single amino acid exchanges in PPR56 (PPR-7TD > TN and PPR-4TN > TD, respectively), we conclude that even such small changes may have significant overall impacts on PPR-type editing factor functionality, possibly reducing (or enhancing) structural flexibility for binding to RNA targets or having an impact on the thermodynamics of the protein-RNA interactions.…”

Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56

“…The contribution of L-type PPRs for target recognition has been investigated previously, ascribing them a role in RNA editing but not in RNA binding [ 61 ]. Notably, the two native targets of PPR56 display different nucleotides opposite of their three central L-type PPRs ( Fig 4 ).…”

Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56

“…The contribution of L-type PPRs for target recognition has been investigated previously, ascribing them a role in RNA editing but not in RNA binding [48]. Notably, the two native targets of PPR56 display different nucleotides opposite of their three central L-type PPRs (Fig.…”