Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

Abstract

ORCID IDs: 0000-0003-0947-2648 (P.K.); 0000-0002-9584-6783 (C.S.B.); 0000-0001-5300-1216 (I.S.)RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.

PPR-SMR protein SOT1 has RNA endonuclease activity

“…Similar to PUF proteins, several PPR proteins have been successfully modified to recognize corresponding RNA targets through simply modifying the critical amino acids of each PPR motif (9,(12)(13)(14)(15)(16)(17). Considering the PPR motifs have been designed to target a specific transcript, if RNA endonuclease activity can be confirmed for the SMR domain of PPR-SMR proteins, the PPR-SMR protein will enable "engineering" of sequence-specific RNA endonucleases, which will have exciting applications for RNA manipulation (27).…”

“…5 B and C). We used a system with multiple fluorescent probes in a single assay (15). RNA13 and RNA13m were 5′ labeled with Cy5 and Cy3, respectively, to produce Cy5 RNA13 and Cy3 RNA13m.…”

“…The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19).…”

“…The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9)(10)(11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).…”

The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript

“…The protein containing an N-terminal 63 His tag was expressed in the E. coli C41 (BL21) strain and purified using Bio-Rad Nuvia resin. REMSA was performed as described previously (Schallenberg-Rüdinger et al, 2013;Kindgren et al, 2015) with a few modifications. Briefly, 10 mL binding buffer consisting of 13 THE (34 mM Tris, 66 mM HEPES, and 0.1 mM EDTA, pH 8.3) with 200 mM NaCl, 5 mM DTT, 5 mg/mL heparin, and 0.1 mg/mL BSA were mixed with 5 mL protein dilution and incubated at room temperature for 10 min.…”