Phase Separation and Disorder-to-Order Transition of Human Brain Expressed X-Linked 3 (hBEX3) in the Presence of Small Fragments of tRNA

Abstract

Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death.

Introduction

The location in chromosomal region Xq22 and early observation of high expression level in the brain [1] coined the name of Brain Expressed X-linked (BEX) protein family. Interestingly, BEX genes' appearance was recent and specific to placentae mammals [2]. This family comprises five protein members in humans whose long regions of intrinsic disorder are conserved across all members [3]. The highly dynamic structural state of intrinsically disordered regions configures these proteins as important points of cell signaling network regulation. For this reason, aberrant function of several proteins containing intrinsically disordered regions is related to cancer development and neurodegeneration [4]. BEX3 is expressed in virtually all human tissues [5]. Expressed Sequence Tag database identified BEX3 gene transcripts overregulated in breast and lung tumors, for instance, while ovary and kidney tumors presented low level of BEX3 transcripts [7] suggesting involvement in different types of cancer. Moreover, the role of BEX3 as a tumor suppressor protein was explored in a xenograft MDA-MB-231 cancer cell model in mice. In this study, tumors formed by cells overexpressing mouse BEX3 (mBEX3) presented reduced size or were even incapable of developing into a sizeable mass compared to control cells [8]. This proapoptotic role of mBEX3 was anticipated by its interaction with the death domain of neurotrophic receptor p75^NTR [9]. However, BEX3 function remains unclear since p75 interaction was not reproduced in human models and, instead, it was observed that rat BEX3 (rBEX3) had pro-survival role through interaction with tyrosine kinase receptor A (TrkA) [8,10,11].

The participation of BEX family in cell signaling through nucleic acid binding has emerged recently. For instance, BEX1 was identified in an RNA-dependent processing complex. This protein showed clear involvement in heart failure progression and specifically binds to proinflammatory AU-rich mRNAs promoting their stabilization [12]. In another example, it was demonstrated that rBEX3 binds to the promoter of trKA, as assessed by chromatin immunoprecipitation assay, and induces its transcription. Moreover, rBEX3 mutant with disrupted nuclear export signal showed strict nuclear localization, consistent with nucleocytoplasmic shuttling. However, it is not known how BEX3 enters the nucleus since it lacks a nuclear localization sequence. The intracellular domain of p75^ICD was speculated as a potential candidate to shuttle BEX3 to the nucleus [11]. Importantly, approximately 90% of proteins involved in transcription regulation have long regions of intrinsic disorder [13]. We wonder whether the nucleic acids' binding property applies to human BEX3 (hBEX3) that shuttles between the nucleus and the cytoplasm and presents intrinsic disorder. In turn, investigations of intrinsically disordered RNA-binding proteins in vitro upon crowding, mimicking the intracellular milieu, provided insightful results in what has been regarded as “protein phase separation: new phase in cell biology” [14]. Liquid–liquid phase separation is the biophysical principle behind assemblage of membraneless organelles, which usually consist of intrinsically disordered proteins and RNA [[15], [16], [17]]. The presence of these distinct macromolecules has a profound impact on condensates formation and function [18]. For instance, RNA can induce protein folding, influence its solubility, and impact on viscoelastic properties of condensates [19,20]. Whereas protein modulates RNA structure, for example, stabilizing hairpin structures and forming a scaffold for RNA storage, sequestration, trafficking, and processing in the interior of condensates [21,22]. Ultimately, aberrant phase separation of aggregation-prone proteins seems to be the key process resulting in dysfunctional aggregates found in neurological diseases [23] and some types of cancer [24,25]. In fact, our group had pointed out that BEX3 forms intracellular granules similar to membraneless organelles as visualized by confocal immunolocalization in human cells [26].

Previously, we characterized mouse BEX3 as a soluble high-order oligomer, the only integrative biophysical study on BEX family reported to date [26]. Here, we produced recombinant hBEX3 in Escherichia coli and noticed that it co-purifies with a small bacterial RNA identified as tRNA-derived fragments. This RNA is responsible for binding-induced folding and impact on liquid–liquid phase separation, as assessed by a range of biophysical and biochemical techniques. We further show that this RNA is base-paired and can modulate hBEX3 oligomerization state, and also influence its phase transitions.