Defining the Link: Characterizing HNRNPH1 Binding to Differentially Structured G-rich Sequences in Childhood Neurodevelopmental Disorders

<p class="p2">Developmental disabilities in children aged 3-17 years increased from 7.40% to 8.56% from 2019 to 2021 (Zablotsky et al., 2023), posing a growing health concern. Recent large-scale genomic studies have begun to identify RNA-binding proteins as major molecular contributors to neurodevelopmental disorders (NDDs). Specifically, mutations in members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, like HNRNPH1, have been implicated as distinct contributors to NDD pathogenesis (Gillentine et al., 2021). While initially identified as a contributor to Bain-type syndromic mental retardation (Pilch et al., 2018), HNRNPH1-related NDD has recently emerged as a distinguishable clinical phenotype characterized by intellectual disability and motor delays (Reichert et al., 2020). <p class="p2">Understanding the molecular basis of HNRNPH1&rsquo;s role in NDDs, therefore, begins with its normal cellular contributions. Like many of its family members, HNRNPH1 is a versatile RNA-binding protein that regulates a variety of co- and post-transcriptional RNA processes (Brownmiller &amp; Caplen, 2023; Zhu et al., 2025). Notably, HNRNPH1 has been shown to regulate alternative splicing by interacting with guanine-rich motifs that fold into canonical RNA G-quadruplex (rG4) structures (Vo et al., 2022). Functionally, HNRNPH1 acts as a non-catalytic destabilizer of its target RNA transcripts, using its qRRM1 and qRRM2 domains to maintain transcripts in an unfolded, favorable splicing state (Vo et al., 2022). Despite these insights into HNRNPH1&rsquo;s function, the mechanisms by which disease-associated mutations disrupt its binding activity remain poorly understood. <p class="p2">This thesis adopts a biophysical approach to characterize the consequences of three disease-associated mutations&mdash;R206W, T286A, and Q415fs*&mdash;located at three distinct domains in HNRNPH1. We obtained all molecular constructs by cloning, expressing, and purifying recombinant HNRNPH1 variants using glutathione-S-transferase (GST) tag cleavage and heparin chromatography. Following purification, fluorescence polarization (FP) assays were employed to quantify the binding affinity between wild-type (WT) and mutant constructs, identifying measurable differences in rG4- and poly-AG-binding affinities. These findings provide the first characterization of how pathogenic mutations in HNRNPH1 alter its binding affinity for G-rich RNA, offering a novel molecular framework for developing innovative therapeutic strategies and improving the well-being of children and families affected by this rare neurodevelopmental disorder.