The role of HNRNPU-locus and circular RNAs in neuronal development and neurodevelopmental disorders

Neuronal development is a complex process regulated by precise gene expression programs involving chromatin organization, epigenetic modifications and RNA processing. Understanding these regulatory mechanisms is essential for elucidating how neural cells acquire and maintain their identity. In this thesis, the roles of RNA-binding protein HNRNPU and circular RNAs in regulating neuronal development were investigated using in vitro cell cultures and cortical organoids as model systems.

In Study I, the focus was on examining the effects of HNRNPU deficiency on neural differentiation, RNA splicing, and 3D genome organization using a 2D in vitro model. HNRNPU deficiency was shown to cause significant disruptions in these processes. Genes that are localized in chromatin regions with differential 3D conformation and genes encoding mRNAs with differential splicing in HNRNPU- deficient cells were associated with neurodevelopmental disorders. Additionally, HNRNPU deficiency was shown to result in increased proportion of neural progenitors within differentiating neural populations. These disrupted trajectories highlight the importance of HNRNPU in maintaining genomic integrity and neural stem cell commitment to differentiation.

The molecular interactome of HNRNPU during neural differentiation was investigated in Study II. Key regulatory networks associated with HNRNPU were identified using ribonucleoprotein immunoprecipitation, mass spectrometry and RNA sequencing. HNRNPU was found to, for example, interact with mammalian SWI/SNF chromatin remodeling complex, bringing new insights into the regulatory mechanisms behind chromatin organization defects seen in relation to HNRNPU deficiency. Identifying HNRNPU binding to mRNAs encoding DNA methylation regulators led to measuring global DNA methylation levels after HNRNPU silencing, revealing significant hypomethylation. This finding linked HNRNPU deficiency to the distinct methylation episignatures reported in individuals with HNRNPU- related neurodevelopmental disorders.

The expression of circular RNAs during neural differentiation was explored in Study III. Key circular RNAs with increasing expression patterns were identified and validated. Interestingly, several circular RNAs were formed from linear mRNAs encoding proteins with synaptic functions, elucidating their possible regulatory roles and contributions to neurodevelopmental processes. Additionally, the exons within these circular RNAs had an increased frequency of genetic variation, and RNA-binding protein SFPQ was identified as a key regulator of these circular RNAs. The findings from this study provide an important resource for further research focusing on the roles of circular RNAs in neuronal development.

In Unpublished results, the effects of different types of mutations affecting the HNRNPU-locus were investigated using a 3D cortical organoid in vitro model. New insights into the early gene expression regulation and epigenetic control exerted by HNRNPU were uncovered during neuronal development through single-cell RNA sequencing and DNA methylation arrays. Preliminary results revealed differences from different types of genetic variants in the HNRNPU-locus.

Collectively, these studies enhance our understanding of the genetic and molecular basis of neurodevelopmental disorders. By elucidating the roles of HNRNPU and circular RNAs in neuronal development, we provide valuable knowledge of the roles of RNA-binding proteins and non-coding RNAs in orchestrating gene expression control during neuronal development.

List of scientific papers

I. Mastropasqua F.#, Oksanen M.#, Soldini C., Alatar S., Arora A., Ballarino R., Molinari M., Agostini F., Poulet A., Watts M.E., Rabkina I., Becker M., Li D., Anderlid B.M., Isaksson J., Lundin Remnelius K., Molsem M., Jacob Y., Falk A., Crosetto N., Bienko M., Santini E., Borgkvist A., Bölte S., Tammimies K. Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis. Biol Open. 2023 Oct 15;12(10):bio060113. https://doi.org/10.1242/bio.060113

II. Oksanen M., Mastropasqua F., Mazan-Mamczarz K., Martindale J.L., Ye X., Arora A., Banskota N., Gorospe M., Tammimies K. Molecular interactome of HNRNPU reveals regulatory networks in neuronal differentiation and DNA methylation. BioRxiv. [Submitted; Preprint]
https://doi.org/10.1101/2025.02.19.638869

III. Watts M.E., Oksanen M., Lejerkrans S., Mastropasqua F., Gorospe M., Tammimies K. Circular RNAs arising from synaptic host genes during human neuronal differentiation are modulated by SFPQ RNA-binding protein. BMC Biol. 2023 May 26;21(1):127. https://doi.org/10.1186/s12915-023-01627-w

# Denotes equal contribution