National Institute for Basic Biology

Cell Biology

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
Technical Assistant:

NAKAYAMA, Kei
MATSUDA, Chisato

The transport of specific mRNAs and local control oftranslation in neuronal dendrites represent an important gene
expression system that provides localized protein synthesis indendrites at just the right time and place. It is believed that
this system controls the location at which neurites willconnect to each other, thereby forming neural networks. Our
main interest is to understand the mechanisms and roles ofmRNA transport and local translation in neuronal dendrites.
It is known that specific mRNAs are recruited into “RNAgranules” in neuronal dendrites. RNA granules aremacromolecular complexes composed mainly of mRNAs,ribosomes and RNA-binding proteins, and mediate thetransport and local translation of their mRNA cargoes in
response to synaptic stimulation (Figure 1). We areresearching RNA granule factors regulating mRNA transportand local translation, their target mRNAs, and themechanisms of localized protein synthesis using mice inorder to better understand its relation to the formation ofsynapses and neural networks, memory, learning, andbehavior.

is associated with defects in fetal brainstem development. To
investigate the role of RNG105 in higher brain functions,e.g., memory and learning, in adult mice, we generated
RNG105 conditional knockout (cKO) mice using the Cre/loxP system. Alpha-CaMKII promoter was used to drive Cre
recombinase since its promoter activity was low during
embryonic stages but elevated after birth in the brain. We
successfully obtained RNG105 cKO mice that grew intoadults (Figure 2A). Expression of RNG105 was markedly
reduced in the cerebrum, especially in the hippocampal
pyramidal neurons of adult cKO mice (Figure 2B). Open
field behavior testing revealed that exploratory activity ofRNG105 cKO mice in a novel environment was not changed
between the first and later trials, although exploratory
activity of wild-type mice was reduced with increasing
number of trials, suggesting that the knockout mice had someproblems in being acclimated to a new environment. We are
currently analyzing learning and memory in the cKO mice.
RNG105 has one paralog, RNG140, which has RNAbinding domains highly conserved with RNG105. RNG105
and RNG140 are localized to different kinds of RNA
granules and their timing of expression is also different:
RNG105 is highly expressed in embryos, but RNG140 ishighly expressed in adults (Shiina and Tokunaga, J. Biol.
Chem. 285, 24260-24269, 2010). We have obtained RNG140
knockout mice and are going to investigate the role ofRNG140 in higher brain functions in adult mice.

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. Knockout mice for RNA granule proteins
We previously identified RNA granule protein 105(RNG105), an RNA-binding protein, as a component ofRNA granules. RNG105 is responsible for mRNA transport
to dendrites, which is required for the encoded proteins to betranslated and function in dendrites for proper networking ofneurons (Shiina et al., J. Neurosci. 30, 12816-12830, 2010).
RNG105 knockout mice exhibit reduced dendritic synapse
formation and reduced dendritic arborization, which resultsin poor development of neuronal networks. The knockout
neonates die soon after birth due to respiratory failure, which14Figure 2. RNG105 conditional knockout (cKO) mice. A, 10-week-old
adult wild-type and RNG105 cKO mice. B, Brain hippocampal slices
from wild-type and RNG105 cKO mice were immunostained with ananti-RNG105 antibody. S, somatic layer; D, dendritic layer ofhippocampal pyramidal neurons. RNG105 was reduced in pyramidalneurons of RNG105 cKO mice.

II. Molecular characterization of the RNA
granule complex
RNA granules are abundantly formed in neurons, but notobserved in many other types of cells. However, theformation of RNA granules is induced by stress such asoxidation and virus infection in the cells. They are called“stress granules” and have common features with neuronal
RNA granules, e.g., they are macromolecular complexes
containing ribosomes and mRNAs, and repress translation ofcargo mRNAs. We previously found that RNG105 was acomponent of not only neuronal RNA granules but also stressgranules, and further that overexpression of RNG105 infibroblastic cells induced the formation of stress granules
which contain mRNAs. Because knowledge about stressgranules helps us in studying neuronal RNA granules, ourresearch includes identification and characterization ofmolecular components of stress granules.
We have identified a novel component of stress granules,
NFAR2. NFAR2 was recruited into RNG105-induced stressgranules and enhanced the assembly of the stress granules(Figure 3). It is known that cellular stress activates masterkinases including PKR, which induces translation repression
and stress granule formation. We found that NFAR2
phosphorylation by PKR enhanced its localization to, and theformation of, stress granules. Furthermore, we found thatNFAR2 was bound by its partner NF45 and antagonized its
effect on stress granules in basal condition, but it overcame
the antagonistic regulation by NF45 when phosphorylated byPKR. Because PKR is reportedly implicated in learning andmemory, these results will provide insights into theregulatory mechanism of neuronal RNA granule formation inthe brain.

Figure 3. NFAR2 is a component of stress granules. A6 cells wereco-transfected with RNG105-monomeric red fluorescent protein 1(mRFP1) and NFAR2-green fluorescent protein (GFP). Expression ofRNG105 induced the formation of cytoplasmic stress granules. NFAR2
was predominantly localized to the nucleus, and it was also recruited intoand enlarged the stress granules (arrows). Scale bar, 10 μm.

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