Molecular interactome of HNRNPU reveals regulatory networks in neuronal differentiation and DNA methylation

Background

Understanding how regulatory networks orchestrate neurodevelopment is a central challenge in neuroscience. This aspect is critical in neurodevelopmental disorders (NDDs), where disruption of key regulators can lead to widespread defects in neuronal differentiation, chromatin remodeling, and RNA processing. One of these regulators is heterogeneous nuclear ribonucleoprotein U (HNRNPU), an RNA-binding protein (RBP) associated with a severe NDD characterized by developmental delay, intellectual disability, autism spectrum disorder, and epilepsy.

HNRNPU has been shown to play an essential regulatory role in RNA metabolism and chromatin structure. In neural cells specifically, it has been demonstrated that HNRNPU influences chromatin compaction, gene expression, and alternative splicing and that disruption of HNRNPU impairs RNA transport in neurons.

Despite this, the interacting partners and specific functions linking HNRNPU’s dysfunction to the neurodevelopmental defects seen in patients remain unclear. Recently, studies have revealed that patients with HNRNPU-related NDDs have distinct DNA methylation signatures, suggesting a post-transcriptional regulatory role that may be involved in its pathogenicity. However, few studies have explored the protein and RNA interaction networks of HNRNPU during neurogenesis. These interactions could be the key to understanding the mechanisms underlying the observed changes in DNA methylation.

In this study, the authors address this knowledge gap by profiling the molecular interactome of HNRNPU across early neural differentiation using iPSC-derived neuroepithelial cells (NES). Through a comprehensive approach, including RNA target identification, protein-protein interaction mapping, and analysis of HNRNPU deficiency, the authors characterize the novel molecular partners and epigenetic changes associated with a loss of HNRNPU, providing insight into the disrupted pathways underlying HNRNPU-related NDDs.

Key findings

HNRNPU interacts with mSWI-SNF complex

To identify the protein-protein interactions (PPI) of HNRNPU, the authors performed a formaldehyde crosslinking immunoprecipitation followed by mass spectrometry. Gene ontology analysis linked HNRNPU to the mammalian SWI-SNF complex, a new connection not previously reported in Biological General Repository for Interaction Datasets (BioGRID) a biological database of protein-protein interactions.

HNRNPU regulates RNA abundance, splicing, stability and translation

Using ribonucleoprotein immunoprecipitation (RIP) and RNA sequencing, the authors identified HNRNPU RNA targets. Some HNRNPU RNA targets exhibit differential abundance or alternative splicing in HNRNPU-deficient human and mouse cells. However, other RNA targets (like ERBB4) are neither differentially abundant nor spliced. Using RT-qPCR, the authors showed increased mRNA stability of non-differently expressed RNA targets. Furthermore, polysome fractionation on a sucrose gradient revealed a shift of these non-differently expressed HNRNPU RNA targets from high-molecular-weight polysomes to lower-molecular-weight polysomes, suggesting a role for HNRNPU in regulating translation.

HNRNPU deficiency alters DNA methylation independently of TET enzymes

This study identified TET1 and TET3, enzymes involved in DNA demethylation, as part of HNRNPU’s RNA targets. To assess global methylation, the authors measured 5-methylcytosine levels. In HNRNPU-deficient cells, global methylation levels decreased at D0 but not at D7. However, TET1 and TET3 showed no differences in expression at D0, and only TET3 significantly increased at D7. These findings suggest that while HNRNPU deficiency affects DNA methylation, this effect is not directly related to its loss of interactions with TET enzymes.

Why highlight this preprint?

This preprint thoroughly investigates the molecular roles of HNRPU, a protein implicated in neurodevelopment, using interactome analysis techniques on multiple models. This allowed the preprint authors to explore direct RNA targets of HNRNPU, which encode neurodevelopmental proteins implicated in pathologies such as epilepsy. In doing so, this study opens new avenues for therapeutic interventions. Also, this paper increases our understanding of HNRNPU and highlights the necessity of investigating previously understood proteins through a new lens (= through techniques not limited to transcriptomics). This paper was interesting to us due to our own work understanding the neuro-molecular mechanisms of disease.The authors do not shy away from asking questions and offering interesting hypotheses based on their results. We hope to apply these qualities to our work. 

Questions for the authors

1. You mention that the experimental conditions may have contributed to the slight changes seen in the ERBB4 mRNA. This is because you saw that the distribution curve of non-target NPM1 mRNA shared the same attributes as those seen in ERBB4. How would you  suggest eliminating these non-HNRNPU-specific changes to confirm their potential role in ERBB4 mRNA translation?
2. This paper proposes that HNRNPU and the mSWI/SNF complex increase the access for transcription factors in early neuronal development. How could this interplay be further assessed in vivo?
3. How does the efficiency of the silencing compare to the haploinsufficiency observed in the patient? How could the different silencing efficiencies change the alteration in methylation seen? Could we see if the other TET enzymes have a similar increase in demethylation as TET3 with an increased silencing efficiency?

Tags: brain development, dna methylation, hnrnpu, interactome, neurodevelopmental disorders, neuronal differentiation

doi: https://doi.org/10.1242/prelights.40844

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