LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
OHASHI, Rie
SOKENDAI Graduate Student: YAMASHITA, Akira
HORIO, Tomoyo
ISHIKURA, Yui
YOSHIDA, Sho

RNA granule assembly, RNA granule factors and their phasebehavior regulating mRNA transport and local translation,
their target mRNAs, and the roles of the locally synthesized
proteins, so we can attain a better understanding of their relationship to the formation of synapses and neural networks,
memory, learning, and behavior.
In addition to the physiological function, RNA granulesare linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar
degeneration (FTLD). The basis of these diseases is thought
to be the accumulation and aggregation of disease-causing
proteins such as FUS and TDP-43 in RNA granules. Thus,
our research also aims to reveal the effects of such protein
aggregation on mRNA transport and local translation.

Figure 1. A model for local translation in neuronal dendrites. SpecificmRNAs are recruited into RNA granules and transported to dendrites.
Translation of the mRNAs is induced locally upon synaptic stimulation,
which modifies local postsynapses to regulate synaptic connection andnetwork formation.

I. Prion-like domain of ILF3 regulates gene
expression and fear memory under chronicstress

RNA granules (green) transported from the cell body (magenta) to dendrites in the hippocampus of the mouse brain.

The transport of specific mRNAs and local control of translation in neuronal dendrites are part of an important gene
expression system that provides dendritic protein synthesis
at exactly the right time and place. It is widely accepted that
this system controls the location at which neurites will stably
connect to each other, thereby forming long-term neuralnetworks and memory. Our main interest is understanding the mechanisms and roles of mRNA transport and local
translation in neuronal dendrites.
Specific mRNAs are recruited into “RNA granules” andtransported to dendrites. RNA granules are membranelessorganelles formed by liquid-liquid phase separation (LLPS)
of RNA-binding proteins and mRNAs, which further recruitother factors such as ribosomes. They mediate the transportof mRNAs to the vicinity of synapses and synaptic stimulation-dependent local translation of the cargo mRNAs (Figure1). We are currently using mice to research the mechanism ofLLPS is generally driven by protein intrinsically disordered regions (IDRs) that do not form three-dimensional
structures. One class of IDR is the prion-like domain(PrLD), whose propensity to form aggregates has been
extensively studied in the context of neurodegenerative
diseases. However, little is known about the physiologicalrelevance of PrLDs in the brain.
We focused on an RNA-binding protein NFAR2, which
has PrLD at the C-terminus. This PrLD is spliced out
in the alternative splicing isoform NFAR1. NFAR1and NFAR2 are synthesized from the Ilf3 gene, andboth possess biochemical activities that they perform incommon such as transcriptional regulation and stressdependent translational inhibition. However, they differ
in that NFAR2 alone can concentrate in the nucleoplasm
and cytoplasmic RNA granules through PrLD. To investigate the physiological relevance of PrLD, we generated
NFAR2ΔPrLD mice, in which a stop codon was introduced into the NFAR2-specific exon. This is considered
an ideal strategy for revealing the physiological role
specific to PrLD, as the splicing isoform without PrLD
is intrinsically present and maintained even after PrLDdeletion.
Deletion of the PrLD lost the nucleoplasmic localizationof NFAR1/2 and affected the genome-wide profiles of21

National Institute for Basic Biology

Cell Biology

mRNA expression and translation in the brain. In particular, PrLD deletion significantly altered chronic restraint
stress (CRS)-induced changes in mRNA expression andtranslation in the amygdala, a brain region associated withemotional events such as anxiety and fear.
Consistent with the effects on CRS-induced changes inmRNA expression and translation, PrLD deletion affected
tolerance of mice to CRS in amygdala-associated learningand memory: conditioned fear memory in wild-typemice was unaffected by CRS, but it was exacerbated in
NFAR2ΔPrLD mice due to CRS. These results indicated that
PrLD of NFAR2 is responsible for the regulation of stressresponsive gene expression and confers stress tolerance infear-associated memory formation, providing insights into
PrLD’s involvement in chronic stress adaptation.

II. Novel regulation of RNA granule dynamics
by pathogenic accumulation of FUS andTDP-43 in RNA granules

w/ TDP-43

w/ FUS

w/o FUS or TDP-43

FUS and TDP-43 are predominantly localized in thenucleus when cells are healthy, but in neurodegenerative
diseases, they translocate to the cytoplasm and accumulate inRNA granules. FUS and TDP-43 prone to aggregate in RNAgranules, which is thought to harden RNA granules, affectthe function of RNA granules, and impair synapse formationand function. However, little is known about the effects of
FUS and TDP-43 on the dynamics of other components ofRNA granules.
We overexpressed disease-related FUS and TDP-43 mutantsin primary cultured neurons from the mouse cerebral cortex
and analyzed their effects on the dynamics of RNA granulecomponents using fluorescence recovery after photobleachRNG105
SunTag (Translation)

RNG105 SunTag Merge

FUS

III. Transport and local translation of mRNAs
encoding the regulators of the small G
protein Arf in neuronal dendrites

We have previously reported that RNG105 knockout in mice
reduces mRNA transport to dendrites and impairs synaptic
long-term potentiation and long-term memory (Nakayama etal., eLife, 2017). Gene ontology enrichment analysis of mRNA
with reduced transport to dendrites in RNG105 knockout mice
revealed that a group of mRNAs encoding regulators of thesmall G protein Arf (Arf GAPs and GEFs) was remarkably
reduced in amount in the dendrites of RNG105 knockout
neurons. Of these mRNAs, Psd mRNA, which encodes an Arf
GEF, was most significantly reduced. In order to clarify thebiological significance of dendritic transport of Psd mRNA,
we are aiming to generate mice in which Psd mRNA transport
can be artificially controlled.
In this study, we identified the 3’UTR of Psd mRNA as acis-element responsible for the transport to dendrites. WhenPsd mRNA lacking the 3’UTR was expressed in mouseprimary cultured neurons, the transport of that mRNA todendrites was reduced. However, it was revealed that the3’UTR was required not only for dendritic transport but also
for translation. Therefore, we narrowed down the responsible
regions for the transport and translation in the 3’UTR, andfound that 5’ half and 3’ half of the 3’UTR were required
for transport and translation, respectively. Based on theseresults, we are proceeding with the generation and analysisof mice in which only the 5’ half of the 3’UTR of Psd mRNAis deleted to reduce only mRNA transport while maintaining
the translation level of Psd mRNA.

Publication List:TDP-43

Figure 2. Accumulation of FUS and TDP-43 reduces translation in/near
RNG105-localizing RNA granules in dendrites. Primary cultured neurons
expressing RNG105-mRFP1 with and without co-expression of FUSSirius or TDP-43-Sirius were analyzed using the SunTag system to detect
translation activity spatiotemporally. The boxed areas containing dendritic RNA granules are magnified in the right panels. The dotted lines
outline the RNA granules. Scale bar, 10 μm.

22ing (FRAP) and cell permeabilization assays (Shiina, J. Biol.
Chem., 2019). FUS and TDP-43 did not affect the dynamics
of most RNA granule components such as Pum2 and Stau1
in RNA granules in dendrites, but did affect the dynamics
of RNG105 (Caprin1). In contrast to the belief model that
FUS and TDP-43 harden RNA granules, the mobility ofRNG105 was significantly increased, resulting in the releaseof RNG105 from RNA granules into the cytoplasm of dendrites. Simultaneously, the amount of mRNAs contained in
RNA granules decreased, and local translation in/near thegranules also decreased (Figure 2). Since RNG105 is responsible for mRNA recruitment to RNA granules and mRNAtransport to dendrites, increased mobility and loss of localization of RNG105 in dendritic RNA granules may affectthese functions, leading to impaired synapse formation andcognitive function.

〔Original papers〕

T., Okimura, K., Shen, J., Guh, Y.-J., Tamai, T.K., Shimada,• Nakayama,A., Minou, S., Okushi, Y., Shimmura, T., Furukawa, Y., Kadofusa,N., Sato, A., Nishimura, T., Tanaka, M., Nakayama, K., Shiina, N.,
Yamamoto, N., Loudon, A.S., Nishiwaki-Ohkawa, T., Shinomiya,A., Nabeshima, T., Nakane, Y., and Yoshimura, T. (2020). Seasonalchanges in NRF2 antioxidant pathway regulates winter depression-likebehavior. Proc. Natl. Acad. Sci. U.S.A. 117, 9594–9603. DOI: 10.1073/pnas.2000278117

K., Shichino, Y., Iwasaki, S., and Shiina, N. (2020).
• Nakazawa,
Implications of RNG140 (caprin2)-mediated translational regulationin eye lens differentiation. J. Biol. Chem. 295, 15029–15044. DOI:
10.1074/jbc.RA120.012715〔Review article〕

R., and Shiina, N. (2020). Cataloguing and selection of mRNAs• Ohashi,localized to dendrites in neurons and regulated by RNA-binding proteinsin RNA granules. Biomolecules 10. DOI: 10.3390/biom10020167

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