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Method of purifying RNA binding protein-RNA complexes

a technology of rna binding protein and complex, which is applied in the field of purifying rna molecules, can solve the problems of difficult identification of rbps targets in a number of autoimmune and genetic diseases

Inactive Publication Date: 2005-10-13
THE ROCKEFELLER UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The targets of RBPs involved in a number of autoimmune and genetic diseases have been difficult to identify, however.

Method used

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  • Method of purifying RNA binding protein-RNA complexes
  • Method of purifying RNA binding protein-RNA complexes
  • Method of purifying RNA binding protein-RNA complexes

Examples

Experimental program
Comparison scheme
Effect test

example 1

CLIP Specifically Isolates Covalently Bound RBP-RNA Complexes

Materials and Experimental Methods

Immunoblot Analysis.

[0307] The following antibodies were used: gephyrin (Transduction laboratories), rabbit Nova antiserum (Buckanovich, R. J. et al, Mol Cell Biol 17, 3194), Hsp90 (Transduction laboratories), rabbit brPTB antiserum (Polydorides, A. D. et al, Proc Natl Acad Sci USA 97, 6350), dimethyl-Histone H3 (Upstate Biotechnology). Antibody to neuronal Hu proteins and Human POMA serum were obtained from paraneoplastic neurologic disease patients. BRPTB antibody was used as previously described (Polydorides AD et al, Proc Natl Acad Sci USA 97: 6350-55). Nova-1 knockout mice.

[0308] Nova-1 knockout mice were previously described (Jensen K B et al, Neuron 25:359-71).

UV Cross-Linking of Mouse Tissue.

[0309] Mouse hindbrain and spinal cord tissue was dissected from 60 postnatal day 4 P8 mice, and rapidly disaggregated in 50-milliliter (ml) polypropylene tubes using a rubber syringe p...

example 2

Monitoring of Linker Ligation and RT-PCR of RNA Molecules

[0333]32P-labeled RNA was size purified after cross-linking and purification from N2A cells using anti-Nova antiserum as described in Example 1. The size of the RNA fragments ranged from 24-150 bases; the modal size of the RNA was approximately 60 bases (FIG. 2A). Purified RNA fragments were ligated to 5′ and 3′ linker oligonucleotides (“linkers”), which added 16 bases to each end of the molecule. The majority of the labeled fragment RNA shifted in size by 32 bases, indicating successful ligation (FIG. 2B). RNA isolated from regions 1 and 2 in FIG. 2B was amplified by RT-PCR with specific primers complementary to the linker sequences (FIG. 2C). The prominent band at 32 bases was the product from the ligation of the two RNA oligonucleotides without insert. The products in (C) were further divided and further amplified by PCR (FIG. 2D).

example 3

CLIP Method using Nova-1 Antisera Enabled the Isolation and Identification of Sequence Fragments Containing Nova-1 Binding Sites

Materials and Experimental Methods

Computer Analysis of Nova CLIP Fragments.

[0334] 3400 control fragments were randomly generated by a computer program from a 200,000 nucleotides long sequence consisting of 66% intronic, 14% exonic and 20% 3′UTR sequences (corresponding to the ratio in Nova CLIP fragments) from random genes on mouse chromosome 1, such that they corresponded in their size to Nova CLIP fragments (with the average size of 71 nucleotides). Another program was made to count the number of particular polynucleotide (up to 20 nucleotides in a row) in each fragment, and calculate the frequency of fragments carrying a certain number of that polynucleotide (for example, YCAY, where Y represents either U or C). An additional program was made to calculate the average frequencies of nucleotides at three positions flanking a particular dinucleotide (CA...

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Abstract

The present invention provides methods for purifying RNA molecules interacting with an RNA binding protein (RBP), and the use of such methods to analyze a gene expression profile of a cell. The invention also provides sequences of RNA molecules that mediate binding to an RBP, proteins encoded by the sequences, a method of identifying the sequences, and the use of the sequences in a screen to identify bioactive molecules. The invention also provides RNA motifs found among the sequences and compounds that bind the RNA motifs. In addition, the invention provides methods of treating diseases associated with a function of an RNA binding protein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Patent Application 60 / 513,183, filed Oct. 23, 2003, which is incorporated herein by referenceFIELD OF THE INVENTION [0002] The present invention provides methods for purifying RNA molecules interacting with an RNA binding protein (RBP), and the use of such methods to analyze a gene expression profile of a cell. The invention also provides sequences of RNA molecules that mediate binding to an RBP, proteins encoded by the sequences, a method of identifying the sequences, and the use of the sequences in a screen to identify bioactive molecules. The invention also provides RNA motifs found among the sequences and compounds that bind the RNA motifs. In addition, the invention provides methods of treating diseases associated with a function of an RNA binding protein. BACKGROUND OF THE INVENTION [0003] RNA binding proteins (RBPs) are frequently targets of human autoimmune or genetic neurol...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34C12Q1/68
CPCC12N15/1006C12Q1/6806C12Q1/6809C12N15/1003
Inventor DARNELL, ROBERTJENSEN, KIRKULE, JERNEJ
Owner THE ROCKEFELLER UNIV
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