Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Elucidating the structural architecture of S-type pentatricopeptide repeat proteins and their RNA editing domains (#129)

Anuradha Mrs Pullakhandam 1 , Charlie Bond 1 , Ian Small 1 2
  1. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia
  2. ARC centre of excellence for plant energy biology, The University of Western Australia, Crawley, WA, Australia

RNA editing is a process of modifying nucleotide bases, resulting in a chemically altered RNA molecule (Ye et al., 2020). In many plants, site-specific editing of RNA transcripts is carried out by pentatricopeptide repeat (PPR) proteins (Okuda et al., 2009). PPR proteins are broadly classified into P type (canonical 35 amino acid motif) and PLS type (P motif, long (L) and S (short) motifs) subfamilies based on their domain structure (Cheng et al., 2016). PLS subfamily of proteins are further classified into various subgroups (PLS (example S type), E1, E2, E+ and DYW) (Cheng et al., 2016). PLS type DYW subgroup proteins are involved in the site-specific binding and editing of RNA (Schallenberg-Rüdinger et al., 2013). To date, more than thousand editing sites have been identified in club moss and hornworts (Lenz and Knoop, 2013), and it has been determined that the cytidine deaminase like conserved C terminal DYW domain in DYW subgroup is responsible for the RNA editing activity (Lenz and Knoop, 2013). While most plant PPR editing proteins are known to edit cytidine to uridine (C to U), very recently reverse editing (uridine to cytidine) has been discovered to occur in ferns, lycophytes, and hornworts (Lenz and Knoop, 2013). To date, there is no experimental structural information for the DYW:KP domain, and the structural architecture, flexibility, and dynamics of the intact PPR-DYW proteins remain unsolved. Therefore, through structural studies we aim to determine the three-dimensional structures of S-type PPR proteins, and DYW-domain and DYW:KP domain containing proteins, to understand the molecular mechanism of action. Furthermore, we also aim to understand the details of RNA binding specificity in S type proteins and how it impacts the conformation of the protein. In order to obtain the required information, wild-type and designer proteins will be expressed and purified, and subjected to appropriate structural methods including computational prediction, X-ray crystallography, cryogenic transmission electronic microscopy (cryo EM) and small angle x-ray scattering (SAXS). RNA-binding and editing assays will be used to evaluate activity of target proteins and rationally designed mutants.

 

  1. Cheng, S., B. Gutmann, X. Zhong, Y. Ye, M.F. Fisher, F. Bai, I. Castleden, Y. Song, B. Song, J. Huang, X. Liu, X. Xu, B.L. Lim, C.S. Bond, S.-M. Yiu, and I. Small. 2016. Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. The Plant Journal. 85:532-547
  2. Lenz, H., and V. Knoop. 2013. PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation. Bioinformatics and Biology Insights. 7:BBI.S11059.
  3. Okuda, K., A.-L. Chateigner-Boutin, T. Nakamura, E. Delannoy, M. Sugita, F. Myouga, R. Motohashi, K. Shinozaki, I. Small, and T. Shikanai. 2009. Pentatricopeptide Repeat Proteins with the DYW Motif Have Distinct Molecular Functions in RNA Editing and RNA Cleavage in <em>Arabidopsis</em> Chloroplasts. The Plant Cell. 21:146.
  4. Challenberg-Rüdinger, M., H. Lenz, M. Polsakiewicz, J.M. Gott, and V. Knoop. 2013. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes. RNA Biology. 10:1549-1556.
  5. Ye, Y., Z. Zhang, Y. Liu, L. Diao, and L. Han. 2020. A Multi-Omics Perspective of Quantitative Trait Loci in Precision Medicine. Trends in Genetics. 36:318-336.