RNA-binding protein

Details RNA-binding protein RNA-binding protein

2001-03-30

2004-09-21

Ketter, James (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100

Reexamination Certificate

active

06794500

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a Synaptotagmin-binding and yet cytoplasmic RNA-binding protein and a gene encoding the protein.
2. Description of the Prior Art
It is known that the release of neurotransmitters from the presynaptic terminal in mammals begins with the fusion of synaptic vesicles with the axon-terminal plasma membrane. The synaptic vesicle protein VAMP1 or 2 and the two presynaptic membrane proteins Syntaxin 1 and SNAP-25 are essential for neurotransmitter release (Scheller, R. H. (1995) Neuron 14, 893-897; Sudhof, T. C. (1995) Nature 375, 645-653; Augustine, G. J. et al. (1996) Annu. Rev. Pharmacol. Toxioccol. 36, 659-701). The formation of a complex among these proteins is the first molecular process for the synaptic vesicle fusion with the above-mentioned plasma membrane.
On the other hand, Synaptotagmin (Syt) is a protein playing an important role in the transport of neurotransmitters and is known to be a family consisting of at least 12 isoforms (Syt-I to Syt-XII) in rat and mouse (Schiavo, G. et al. (1998) Biochem. Biophys. Res. Commun. 248, 1-8; Li, C. et al. (1995) Nature 375, 594-599). Among these isoforms, Syt-I is present on the synaptic vesicle membrane as an integral protein spanning the membrane once. This protein possesses two domains (termed C2A and C2B) homologous to the C2 regulatory region of protein kinase C (Sudhof, T. and Rizo, J. (1996) Neuron 17, 379-388; Schiavo, G. et al., (1998) Biochem. Biophys. Res. Commun. 248, 1-8). Syt-I is one of the proteins involved in neurotransmission and functions via the following mechanism. The C2B domain of Syt-I on synaptic vesicles fuses with the plasma membrane (presynaptic membrane) to thereby open a fusion pore in the synaptic vesicle membrane. Then, neurotransmitters in the synaptic vesicle are transported through the pore.
The present inventor has found that inositol 1,3,4,5-tetrakisphosphate (IP4) binds to the C2B domain of Syt-I, -II, -IV, -VI through -IX and -XI (Fukuda, M. et al. (1994) J. Biol. Chem. 269, 29026-29211; Ibata, K. et al. (1998) J. Biol. Chem. 273, 12267-12273), and demonstrated that inositol high polyphosphate series (IHPS; IP4 and IP6) play an important role in the regulation of Syts-regulated vesicle trafficking (Fukuda, M. and Mikoshiba, K. (1997) BioEssays 19, 593-603). For example, the binding of an inositol high polyphosphate to the C2B domain of Syt inhibits the step of fusion between synaptic vesicles and the presynaptic membrane (Fukuda, M. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10703-10707; Mochida, S. et al. (1997) Neuroscience 77, 937-943; Ohara-Imaizumi, M. et al. (1997) Proc. Natl. Acad. Sci. USA 92, 10708-10712). Thus, the C2B domain and the presynaptic membrane are separated from each other by the presence of IP4, which results in the inhibition of neurotransmission.
Thus, it is believed that various substances are involved in the interaction of synaptic vesicles with the presynaptic membrane.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the invention to provide a Synaptotagmin-binding and yet cytoplasmic RNA-binding protein.
Toward the solution of the above problem, the present inventor has analyzed those proteins capable of binding to Synaptotagmin and succeeded in isolating a protein which binds to Synaptotagmin and yet can bind to cytoplasmic RNA. Thus, the present invention has been achieved.
The present invention relates to the following inventions:
(1) A recombinant protein selected from the group consisting of the following (a) and (b):
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2
(b) a protein which comprises the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has RNA binding activity.
(2) A recombinant protein selected from the group consisting of the following (c) and (d):
(c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4
(d) a protein which comprises the amino acid sequence as shown in SEQ ID NO: 4 having deletion, substitution or addition of one or several amino acids and which has RNA binding activity.
(3) A gene encoding a protein selected from the group consisting of the following (a) and (b):
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2
(b) a protein which comprises the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has RNA binding activity.
(4) A gene encoding a protein selected from the group consisting of the following (c) and (d):
(c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4
(d) a protein which comprises the amino acid sequence as shown in SEQ ID NO: 4 having deletion, substitution or addition of one or several amino acids and which has RNA binding activity.
(5) A gene comprising a DNA selected from the group consisting of the following (e) and (f):
(e) a DNA comprising the nucleotide sequence as shown in SEQ ID NO: 1
(f) a DNA which hybridizes to the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and which encodes a protein having RNA binding activity.
(6) A gene consisting of a DNA selected from the group consisting of the following (g) and (h):
(g) a DNA comprising a nucleotide sequence spanning from position 154 to position 1836 of SEQ ID NO: 3
(h) a DNA which hybridizes to a DNA comprising a nucleotide sequence spanning from position 154 to position 1836 of SEQ ID NO: 3 and which encodes a protein having RNA binding activity.
(7) A recombinant vector comprising the gene.
(8) A transformant comprising the recombinant vector.
(9) A method of producing an RNA-binding protein comprising culturing the above transformant and recovering the RNA-binding protein from the resultant culture.
(10) An antibody against the protein.
(11) A pharmaceutical composition for regulating neuronal functions, comprising the protein as an active ingredient.
(12) A therapeutic agent for neurological diseases comprising the protein as an active ingredient.
(13) A reagent for detecting a Synaptotagmin-binding and yet RNA-binding protein, comprising an antibody against the protein.
(14) A reagent for detecting Synaptotagmin, comprising the protein and/or an antibody against the protein.


REFERENCES:
Mizutani et al. SYNCRIP, a cytoplasmic counterpart of herogenous nuclear ribonucleoprotein R, interacts with ubiquitous sunaptotagmin isoforms J. Biol. Chem. vol. 275 No. 13 2000, pp 9823-9831.*
Accession No.: AB035725 GenBank Mar. 31, 2000.*
Akihiro Mizutani et al. “SYNCRIP, a Cytoplasmic Counterpart of Heterogeneous Nuclear Ribonucleoprotein R, Interacts with Ubiquitous Synaptotagmin Isoforms.”, The Journal of Biological Chemistry, vol. 275, No. 13, pp. 9823-9831, Mar. 31, 2000, Laboratory for Developmental Neurobiology and for Molecular Neurogenesis, the Brain Science Institute, the Institute of Physical and Chemical Research (RIKEN) . . . Japan.

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