A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platfo...

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Bibliographic Details
Published in:Molecular cell Vol. 74; no. 5; p. 966
Main Authors: Jarmoskaite, Inga, Denny, Sarah K, Vaidyanathan, Pavanapuresan P, Becker, Winston R, Andreasson, Johan O L, Layton, Curtis J, Kappel, Kalli, Shivashankar, Varun, Sreenivasan, Raashi, Das, Rhiju, Greenleaf, William J, Herschlag, Daniel
Format: Journal Article
Language:English
Published: United States 06.06.2019
Subjects:
Amino Acid Sequence - genetics
Humans
Kinetics
Nucleic Acid Conformation
Protein Binding - genetics
Ribosomes - chemistry
Ribosomes - genetics
RNA, Messenger - genetics
RNA-Binding Proteins - chemistry
RNA-Binding Proteins - genetics
high-throughput biophysics
PUF proteins
post-transcriptional regulation
thermodynamics
Pumilio
eCLIP
RNA binding proteins
ISSN:1097-4164, 1097-4164
Online Access:Get more information
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Summary:High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.
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ISSN:1097-4164
1097-4164
DOI:10.1016/j.molcel.2019.04.012