News Release

Identification of a novel nuclear structure composed of RNA and RNAbinding proteins to conduct RNA splicing
Key Points•RNA binding proteins (RBPs) form meshworks in the nucleus to drive RNAsplicing•Charged/uncharged intrinsically disordered regions (IDRs) in the RBPs
promote mutual exclusion of their meshworks on RNA•Impaired formation of RBP meshworks leads to aberrant splicing associatedwith neurodegeneration

Summary
In eukaryotes, RNA processing, including RNA splicing, occurs primarilyin the nucleus to produce mature mRNA, which is essential for protein synthesis.

Splicing is a complex process catalyzed by a multi-megadalton machinery,
the spliceosome. It comprises five small nuclear ribonucleoproteins (UsnRNPs) and hundreds of proteins, including core spliceosomal RNA-binding
proteins (RBPs) and accessory RBPs. However, specific nuclear structuresassociated with the spatiotemporal regulation of splicing remain elusive.
Here, we found that core RBPs and accessory RBPs form two distinct

meshworks that are adjacently but distinctly distributed throughout thenucleus. Core RBPs and accessory RBPs harbor charged and unchargedintrinsically disordered regions (IDRs), respectively, which mutually exclude

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each other during their condensation. In the nucleus, the binding of these RBPs
to nascent RNA facilitates the formation of fibrous structures. Cross-linking
and immunoprecipitation (CLIP)-seq and RNA-seq analyses revealed that thefibers of core RBPs and accessory RPBs compete for space occupancy on
nascent RNA to drive splicing. Furthermore, the optogenetic enhancement of
the RBP meshwork resulted in aberrant splicing of genes associated withneurodegenerative diseases. We identified the spatial organization of RBP
networks to regulate RNA splicing.
This study was published online by the Molecular Cell on July 24, 2024.
Research BackgroundThe eukaryotic nucleus is an intricated structure where DNA, RNA, andproteins are organized in a spatiotemporal manner. Following transcription ofpre-mRNA, RNA processing, such as RNA splicing, 5’ capping. 3’ end processing,and RNA modification, occur in the nucleus to produce mature mRNA. Then, themRNA is transported to the cytoplasm for translation in the ribosome.
Importantly, RNA processing increases proteome diversity by generating
multiple mRNA isoforms from a gene sequence encoded in DNA.
Splicing is a complex process facilitated by the spliceosome, a large
machinery composed of small nuclear ribonucleoproteins and hundreds ofproteins, including core RNA-binding proteins (core RBPs). Accessory RBPs
modulate spliceosome activity by binding to specific sites in pre-mRNA.
Although splicing is conducted in a spatiotemporal manner, specific nuclear
structures associated with the spatiotemporal regulation of splicing remain
elusive.
Research Results
We analyzed the subcellular distribution of RBPs using super-resolution
microscopy strategies, of which resolutions are beyond the optical diffraction
limit. We observed that at least 13 RBPs were distributed in meshworks
throughout the nuclei of cells in various human and mouse tissues (Fig. 1).
Although core RBPs and accessory RBPs form distinct meshworks, they wereadjacent and intertwined.

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Live imaging analysis of cells expressing EGFP-tagged MATR3 (accessory RBP)
showed that transcription arrest by DRB treatment dissipated the MATR3
meshwork and formed large droplets (Fig. 2). Upon the removal of DRB, thelarge droplets reverted to small patches to form meshworks, suggesting therequirement of nascent RNA for the formation of the RBP meshwork.

In silico analysis of the amino acid sequences of RBPs revealed that core RBPs

and accessory RBPs harbor distinct classes of charged uncharged andcharged intrinsically disordered regions (IDRs), respectively (Fig. 3, left).

In vitro analysis with the recombinant proteins and in cellulo analysis
with overexpression of the IDRs showed that the charged and uncharged
IDRs in RBPs form separating condensates (Fig. 3, right).

3 / 6We next performed cross-linking and immunoprecipitation (CLIP)-seq tounderstand the role of RBP meshworks in RNA binding. Our analysis
demonstrated that the local accumulation of the accessory RBP with anuncharged IDR (MATR3) prevents the binding of the core RBP with a charged
IDR (SF3B1), but not of the accessory RBP with an uncharged IDR (PTBP1), topre-mRNA (Fig. 4). These results suggest that the meshworks of core RBPs andaccessory RBPs compete for spatial occupancy on pre-mRNA.

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Finally, we investigated the role of meshwork formation in alternative splicing
regulation using an optogenetic system. We confirmed that blue light irradiationinduced the condensation of overexpressed MATR3, coarsening its meshwork(Fig. 5A). RNA-seq analysis showed that the artificial coarsening of the MATR3
meshwork represses splicing of genes associated with neurodegenerative
diseases (Fig. 3 BC). In addition, we observed that the introduction of an ALScausative mutation of MATR3 into the genomic DNA of N2A cells by CISPR/Cas9system caused the fragmentation of the MATR3 meshwork in these cells.

Research Summary and Future Perspective
We demonstrate that RBPs form meshworks that spread throughout thenucleus to drive RNA splicing. Core RBPs and accessory RBPs constitute twodistinct meshworks that are adjacently but distinctly distributed, and mutually
exclusive on pre-mRNA. The separation of the charged and uncharged IDRs inthese RBPs achieves mutual exclusion. Our analysis revealed that the spatialorganization of RBP networks is a functional regulator of RNA splicing.
We observed that the impaired formation of RBP meshworks causes
aberrant splicing associated with neurodegeneration. Furthermore, we showedthat the ALS causative mutation in the IDR affects the meshwork formation. We
hope that our work will contribute to the understanding of the pathogenesis ofneurodegenerative diseases.
Publication
Title: Blending and separating dynamics of RNA-binding proteins developarchitectural splicing networks spreading throughout the nucleus
Authors: Akio Masuda,1,3 Takaaki Okamoto,1,3 Toshihiko Kawachi,1 Jun-ichi
Takeda,1 Tomonari Hamaguchi,1 Kinji Ohno1,2

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Affiliation:
1Division of Neurogenetics, Center for Neurological Diseases and Cancer,

Nagoya University Graduate School of Medicine, Nagoya, Japan
2

Graduate School of Nutritional Sciences, Nagoya University of Arts andSciences, Nisshin, Japan
3

Equal contribution

Journal： Molecular Cell
DOI: 10.1016/j.molcel.2024.07.001

Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Mol_240725.pdf

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