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rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations.

Authors
  • Garriga-Canut, Mireia1
  • Yang, Jae-Seong2
  • Preusser, Friedrich1
  • Speroni, Silvia1
  • Gili, Maria1
  • Maurer, Sebastian P3
  • 1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain. , (Spain)
  • 2 Centre de Recerca en Agrigenòmica, Consortium CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, 08193 Barcelona, Spain. , (Spain)
  • 3 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain. Electronic address: [email protected] , (Spain)
Type
Published Article
Journal
Methods
Publisher
Elsevier
Publication Date
Jun 01, 2020
Volume
178
Pages
19–32
Identifiers
DOI: 10.1016/j.ymeth.2019.09.002
PMID: 31493518
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Understanding which proteins and RNAs directly interact is crucial for revealing cellular mechanisms of gene regulation. Efficient methods allowing to detect RNA-protein interactions and dissect the underlying molecular origin for RNA-binding protein (RBP) specificity are in high demand. The recently developed recombination-Y3H screening (rec-Y3H) enabled many-by-many detection of interactions between pools of proteins and RNA fragments for the first time. Here, we test different conditions for protein-RNA interaction selection during rec-Y3H screening and provide information on the screen performance in several selection media. We further show that rec-Y3H can detect the nucleotide and amino acid sequence determinants of protein-RNA interactions by mutating residues of interacting proteins and RNAs simultaneously. We envision that systematic RNA-protein interface mutation screening will be useful to understand the molecular origin of RBP selectivity and to engineer RBPs with targeted specificities in the future. Copyright © 2019 Elsevier Inc. All rights reserved.

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