Molecular principles underlying dual RNA specificity in the Drosophila SNF protein

Gert Weber,Kathleen B Hall,Gregory T Dekoster,Markus C Wahl,Nicole Holton

doi:10.1038/s41467-018-04561-6

Abstract

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A′ protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A′ or U2 stem-loop IV and U2A′, SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A′ immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A′ can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts.

Highlights

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity
Analysis of binding thermodynamics revealed that Drosophila U2A′ cooperatively restores highaffinity binding of dSNFRRM1 to Drosophila U2SLIV (dU2SLIV), under conditions where it does not influence dSNFRRM1 binding to dU1SLII17, it can associate with the latter complex[16]
With the characterization of dSNFRRM1 dynamics alone and in complexes by NMR spectroscopy and targeted mutagenesis combined with binding studies, we delineated how the intrinsic dSNFRRM1 RNA-binding capacity and specificity are fine-tuned by networks of intra-molecular interactions that modulate dSNFRRM1 dynamics, and how the auxiliary dU2A′ protein can capitalize on intrinsic dSNFRRM1 flexibility to gear dSNFRRM1 binding to dU2SLIV

Summary

Introduction

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A′ protein. Jawed vertebrates use U1A to bind stem-loop II of U1 snRNA (U1SLII) and U2B′′ in conjunction with the leucine-rich repeat (LRR) protein U2A′ to bind SLIV of U2 snRNA (U2SLIV)[10]. Both U1A and U2B′′ employ an N-terminal RNA recognition motif (RRM) to recognize their RNA targets. With the characterization of dSNFRRM1 dynamics alone and in complexes by NMR spectroscopy and targeted mutagenesis combined with binding studies, we delineated how the intrinsic dSNFRRM1 RNA-binding capacity and specificity are fine-tuned by networks of intra-molecular interactions that modulate dSNFRRM1 dynamics, and how the auxiliary dU2A′ protein can capitalize on intrinsic dSNFRRM1 flexibility to gear dSNFRRM1 binding to dU2SLIV

Methods

Results

Conclusion