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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Miranda RG,McDermott JJ,Barkan A

doi

10.1093/nar/gkx1288

subject

Has Abstract

pub_date

2018-03-16 00:00:00

pages

2613-2623

issue

5

eissn

0305-1048

issn

1362-4962

pii

4780162

journal_volume

46

pub_type

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