The First Crystal Structure of the UP1 Domain of hnRNP A1 Bound to RNA Reveals a New Look for an Old RNA Binding Protein

Abstract

The hnRNP A1 protein is a multifunctional RNA binding protein implicated in a wide range of biological functions. Mechanisms and putative hnRNP A1-RNA interactions have been inferred primarily from the crystal structure of its UP1 domain bound to ssDNA. RNA stem loops represent an important class of known hnRNP A1 targets, yet little is known about the structural basis of hnRNP A1-RNA recognition. Here, we report the first high-resolution structure (1.92 Å) of UP1 bound to a 5′-AGU-3′ trinucleotide that resembles sequence elements of several native hnRNP A1 RNA stem loop targets. UP1 interacts specifically with the AG dinucleotide sequence via a “nucleobase pocket” formed by the beta sheet surface of RRM1 and the inter-RRM linker; RRM2 does not contact the RNA. The inter-RRM linker forms the lid of the nucleobase pocket and we show using structure-guided mutagenesis that the conserved salt bridge interactions (R75:D155 and R88:D157) on the alpha helical side of the RNA binding surface stabilize the linker in a geometry poised to bind RNA. We further investigated the structural basis of UP1 binding HIV SL3ESS3 by determining a SAXS-scored structural model of the complex. UP1 docks on the apical loop of SL3ESS3 using its RRM1 domain and inter-RRM linker only. The biophysical implications of the structural model were tested by measuring kinetic binding parameters, where mutations introduced within the apical loop reduce binding affinities by slowing down the rate of complex formation. Collectively, the data presented here provide the first insights into hnRNP A1-RNA interactions.

Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

“…Decreases in k on, app values were previously determined as the predominant mechanism that contributes to weaker affinities for cytosine-substituted SL3 ESS3 constructs, and similar observations have recently been made for other protein-RNA systems (13,25,27). Thus, for some SL3 ESS3 variants, specificity is driven, in large part, by the frequency with which UP1 collides productively with a cognate RNA that minimally exposes a conformationally flexible 5′-AG-3′ motif.…”

“…We previously showed UP1 interacts with native SL3 ESS3 through its RRM1 domain and inter-RRM linker to form a 1:1 complex (25). Although capable of binding RNA, RRM2 is not considered to be the preferred high-affinity binding surface.…”

“…Transcriptome-wide studies of functional hnRNP A1 binding sites using motif finder algorithms further indicate specificity for a 5′-UAG-3′ motif (11,24). In previous work, we determined a small angle X-ray scattering (SAXS) model of UP1 in complex with the HIV ESS3 stem loop (SL3 ESS3 ), which was guided by a 1.92-Å crystal structure of UP1 bound to a 5′-AGU-3′ oligomer (25). The structure indicates that RRM1 and the inter-RRM linker fold to form a nucleobase pocket that specifically recognizes 5′-AG-3′, which is part of the 7-nt SL3 ESS3 apical loop (5′-GAUUAGU-3′) (Fig.…”

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

“…Thus, this work reveals that HIV-1 uses a phylogenetically conserved RNA structure to recruit the host hnRNP A1 protein upstream to acceptor site A7. When combined with our previous studies on ESS3 (14,16), the mechanism that begins to emerge is one wherein the RNA structure functions as a scaffold to direct the assembly of a functional protein-RNA complex that occludes site A7. This work puts us one step closer to determining the architecture of the intact complex.…”

“…UP1 binds site-specifically to an AG dinucleotide motif of the SL3 ESS3 apical loop (16). Secondary structure probing of the isolated ssA7 locus revealed that the ISS element folds into a stem loop structure with an exposed AGUGA apical loop (Fig.…”