RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions.

Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that offer a promising scaffold for the engineering of proteins to bind specified RNAs. PPR tracts bind RNA in a modular 1-repeat, 1-nucleotide fashion. An amino acid code specifying the bound nucleotide has been elucidated. However, this code does not fully explain the sequence specificity of native PPR proteins. Furthermore, it does not address nuances such as the contribution toward binding affinity of various repeat-nucleotide pairs or the impact of mismatches between a repeat and aligning nucleotide. We used an in vitro bind-n-seq approach to describe the population of sequences bound by four artificial PPR proteins built from consensus scaffolds. The specificity of these proteins can be accounted for by canonical code-based nucleotide recognition. The results show, however, that interactions near the 3'-end of binding sites make less contribution to binding affinity than do those near the 5'-end, that proteins with 11 and 14 repeats exhibit similar affinity for their intended targets but 14-repeats are more permissive for mismatches, and that purine-binding repeats are less tolerant of transversion mismatches than are pyrimidine-binding motifs. These findings have implications for mechanisms that establish PPR-RNA interactions and for optimizing PPR design to minimize off-target interactions.

Unexpected functional versatility of the pentatricopeptide repeat proteins PGR3, PPR5 and PPR10.

Pentatricopeptide repeat (PPR) proteins are a large family of helical repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3'-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3'-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.

RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.

Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design.

The PPR-SMR protein PPR53 enhances the stability and translation of specific chloroplast RNAs in maize.

Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind RNA and influence gene expression in mitochondria and chloroplasts. Several PPR proteins in plants harbor a carboxy-terminal small-MutS-related (SMR) domain, but the functions of the SMR appendage are unknown. To address this issue, we studied a maize PPR-SMR protein denoted PPR53 (GRMZM2G438524), which is orthologous to the Arabidopsis protein SOT1 (AT5G46580). Null ppr53 alleles condition a chlorotic, seedling-lethal phenotype and a reduction in plastid ribosome content. Plastome-wide transcriptome and translatome analyses revealed strong defects in the expression of the ndhA and rrn23 genes, which were superimposed on secondary effects resulting from a decrease in plastid ribosome content. Transcripts with processed 5'-ends mapping approximately 70 nucleotides upstream of rrn23 and ndhA are absent in ppr53 mutants, and the translational efficiency of the residual ndhA mRNAs is reduced. Recombinant PPR53 binds with high affinity and specificity to the 5' proximal region of the PPR53-dependent 23S rRNA, suggesting that PPR53 protects this RNA via a barrier mechanism similar to that described for several PPR proteins lacking SMR motifs. However, recombinant PPR53 did not bind with high affinity to the ndhA 5' untranslated region, suggesting that PPR53's RNA-stabilization and translation-enhancing effects at the ndhA locus involve the participation of other factors.

WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis.

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.