Endribonuclease-containing composition, protein-containing composition, use of polynucleotides, use of expression vectors, use of transformants, method for producing RNA degradation products, and method for cleaving RNA.

Abstract

To provide a novel endoribonuclease having the capacity for sequence-specific RNA cleavage.SOLUTION: This endoribonuclease comprises the amino acid sequence of (a) or (b) below and hydrolyzes the phosphodiester bond between U and A in 5'-UACU-3' or 5'-UACG-3' included in RNA. (a) An amino acid sequence consisting of a specific sequence. (b) An amino acid sequence having 50% or greater sequence identity with the amino acid sequence.SELECTED DRAWING: Figure 4

Claims

[Claim 1] An endribonuclease-containing composition for hydrolyzing the phosphate diester bond between U and A in 5'-UACU-3' or 5'-UACG-3' contained in RNA, comprising an endribonuclease having the amino acid sequence of (a) or (b) below. (a) Amino acid sequence shown in Sequence ID No. 1 (b) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 [Claim 2] A protein-containing composition for inhibiting endoribonuclease activity by binding to an endoribonuclease having the amino acid sequence of (e) or (f) below, comprising a protein having an amino acid sequence selected from the group consisting of (a), (b), (c), and (d) below. (a) Amino acid sequence shown in Sequence ID No. 3 (b) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 3 (c) Amino acid sequence shown in Sequence ID No. 4 (d) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 4 (e) Amino acid sequence shown in Sequence ID No. 1 (f) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 [Claim 3] Use of a polynucleotide comprising a base sequence encoding the endribonuclease for the production of the endribonuclease-containing composition according to claim 1. [Claim 4] Use of a polynucleotide comprising a base sequence encoding the protein for the production of the protein-containing composition according to claim 2. [Claim 5] Use of an expression vector comprising a polynucleotide consisting of a base sequence encoding the endribonuclease for the production of the endribonuclease-containing composition according to claim 1. [Claim 6] Use of an expression vector comprising a polynucleotide consisting of a base sequence encoding the protein for the production of the protein-containing composition according to claim 2. [Claim 7] Use of a transformant having an expression vector comprising a polynucleotide comprising a base sequence encoding the endribonuclease, for the production of the endribonuclease-containing composition according to claim 1. [Claim 8] Use of a transformant having an expression vector comprising a polynucleotide comprising a base sequence encoding the protein, for the production of the protein-containing composition according to Claim 2. [Claim 9] Use of an expression vector for the production of an endribonuclease-containing composition according to Claim 1, The expression vector is A polynucleotide consisting of a base sequence encoding the aforementioned endribonuclease, A polynucleotide comprising a base sequence encoding a protein consisting of an amino acid sequence selected from the group consisting of (c), (d), (e), and (f) below, Includes, use. (c) Amino acid sequence shown in Sequence ID No. 3 (d) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 3 (e) Amino acid sequence shown in Sequence ID No. 4 (f) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 4 [Claim 10] Use of a transformant for the production of the endoribonuclease-containing composition according to claim 1, The transformed body is A polynucleotide consisting of a base sequence encoding the aforementioned endribonuclease, A polynucleotide comprising a base sequence encoding a protein consisting of an amino acid sequence selected from the group consisting of (c), (d), (e), and (f) below, Uses an expression vector that includes the following. (c) Amino acid sequence shown in Sequence ID No. 3 (d) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 3 (e) Amino acid sequence shown in Sequence ID No. 4 (f) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 4 [Claim 11] Use of a transformant for the production of the endribonuclease-containing composition according to Claim 1, The transformed body is An expression vector comprising a polynucleotide consisting of a base sequence encoding the endribonuclease, A gene encoding an RNA polymerase that is expressed in the presence of an expression inducer and expresses the endribonuclease encoded by the polynucleotide, A gene encoding lysozyme that inhibits the RNA polymerase expressed in the absence of the expression inducer, To have, to use. [Claim 12] The present invention includes a degradation step in which the endribonuclease-containing composition described in claim 1 is applied to RNA in vitro, thereby degrading the RNA by the endribonuclease. A method for producing RNA degradation products. [Claim 13] The method further includes a stopping step in which a protein is bound to the endribonuclease to stop the degradation of the RNA, The aforementioned protein is The amino acid sequence consists of an amino acid sequence selected from the group consisting of (c), (d), (e), and (f) below. A method for producing RNA degradation products according to claim 12. (c) Amino acid sequence shown in Sequence ID No. 3 (d) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 3 (e) Amino acid sequence shown in Sequence ID No. 4 (f) Amino acid sequences having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 4 [Claim 14] The present invention comprises a cleavage step in which the endoribonuclease-containing composition described in claim 1 is used in vitro to hydrolyze the phosphate diester bond between U and A in 5'-UACU-3' or 5'-UACG-3' contained in RNA using the endoribonuclease, Methods for cleaving RNA.

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