Methods for identifying functionally related genes and drug targets

a functionally related gene and drug target technology, applied in the field of methods for identifying functionally related genes and drug targets, can solve problems such as the inability of the vitro method to refl

Inactive Publication Date: 2006-11-16
RIBONOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0007] The invention provides methods and compositions that are used to identify, utilize, and characterize mRNP complexes to identify functionally related gene products that are coordinately expressed and associated with a particular mRNP complex. The gene products associated with a particular mRNP complex are classified into biologically relevant subsets on the basis of structural and / or functional relationships. These gene products, including mRNAs, RNA binding proteins, other mRNP complex-associated proteins, may participate in a particular biological pathway, such as an enzyme pathway, or may participate in other cellular event or pathology, such as tumor growth, apoptosis, differentiation, aging, or cell toxicity, for example. The functionally and structurally related gene products that are identified and quantified create a ribonomic profile for the cell or population of cells. This ribonomic profile provides a snapshot of the flow of genetic information at a given time in the life of the cell or cell population, in a normal or diseased state, or in response to an environmental influence or drug. The ribonomic profile is used as a diagnostic marker for disease or other cellular event and to rapidly identify therapeutic targets and therapeutics that alter the expression of one or more of the mRNP complex-associated gene products. The identified gene products themselves are also used as diagnostic and therapeutic indicators.

Problems solved by technology

However, not all mRNAs that bind to an RNA binding protein possess an ARE or other common regulatory element.
However, these in vitro methods do not reflect the dynamic nature of mRNA association with mRNP complexes in vivo, which changes in response to intra- and inter-cellular signaling events.

Method used

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  • Methods for identifying functionally related genes and drug targets
  • Methods for identifying functionally related genes and drug targets
  • Methods for identifying functionally related genes and drug targets

Examples

Experimental program
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Effect test

example 1

RNase Protection in a Multiprobe System

[0093] A multiprobe RNase protection assay was used to rapidly optimize the immunoprecipitation of several endogenous mRNP complexes containing different RNA binding proteins. In the multiprobe system, many mRNAs associated with an mRNP complex can be assayed in a single lane of a polyacrylamide gel.

[0094] Cell Culture and Transformation. Murine P19 embryonal carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.) and maintained in monolayer culture using α-Minimum Essential Medium Eagle (MEM) without phenol red (Gibco BRL 41061-0291) (Invitrogen, Carlsbad, Calif.) supplemented with 7.5% Bovine Calf Serum (BCS), 2.5% Fetal Bovine Serum (FBS) (Hyclone, Logan, Vt.) and 100 U of Penicillin / Streptomycin. Cells were grown in tissue culture flasks or plates that had been pre-coated with 0.1% gelatin (Sigma Chemicals, St. Louis, Mo.) and removed prior to use. Monolayer cell cultures were maintained in 5% CO2 at ...

example 2

Identification of mRNA Subsets Associated with RNA Binding Proteins En Masse Using cDNA Arrays

[0101] A cDNA array (FIG. 5) was used to detect an mRNA subset without amplification or iterative selection.

[0102] Antibodies. Monoclonal anti-gene 10 (g10) antibodies and polyclonal sera reactive with the proteins were produced as previously described. Antibody against 5′ cap binding protein (elF-4E) was obtained from Transduction Laboratories (San Diego, Calif.). Antibodies reactive with Poly A-binding protein (PABP) were obtained from McGill University (Canada).

[0103] Cell Culture and Differentiation. Transgenic cells were prepared as described in Example 1. Cells were treated with retinoic acid (RA) to induce neuronal differentiation as follows: 5×105 p19 cells were placed on a 60 mm petri dish (Fisher Scientific, Pittsburgh, Pa., Number 8-757-13A) with 0.5 μM RA (Sigma Chemicals, St. Louis, Mo., Number R2625). After two days, 25% of the cells that had formed into clumps were removed...

example 3

Alterations in mRNP Complexes in Response to Retinoic Acid

[0110] Because HuB is predominantly a neuronal protein believed to play a role in regulating neuronal differentiation, studies were conducted to investigate whether the mRNA population found in HuB mRNP complexes changes in response to RA, a chemical inducer of neuronal differentiation. HuB-transfected P19 cells were treated with RA to induce the onset of neuronal differentiation, HuB mRNP complexes were immunoprecipitated, and then associated mRNAs were identified on cDNA arrays as described in Examples 1 and 2. Comparison of the mRNA profiles extracted from the HuB mRNP complexes before and after RA treatment revealed that eighteen mRNAs were either exclusively present or greatly enriched (four-fold or greater) in RA-treated HuB mRNPs (FIGS. 7A and 7B). In addition, three mRNAs (T-lymphocyte activated protein, DNA-binding protein SATB1, and HSP84) decreased in abundance by four-fold or greater in response to RA treatment (...

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Abstract

The identification and evaluation of mRNA and protein targets associated with mRNP complexes and implicated in the expression of proteins involved in common physiological pathways is described. Effective targets are useful for treating a disease, condition or disorder associated with the physiological pathway.

Description

RELATED APPLICATIONS [0001] This application is a divisional of U.S. patent application Ser. No. 10 / 309,788. filed on Dec. 4. 2002. which is a continuation-in-part of U.S. patent application Ser. No. 09 / 750,401, filed on Dec. 28, 2000, which claims the benefit of U.S. Provisional Application Ser. No. 60 / 173,338, filed Dec. 28, 1999, both of which are hereby incorporated by reference in their entirety.GOVERNMENT SUPPORT [0002] This invention was made with government support under grant number R01 CA79907 from the National Institutes of Health. The United States Government has certain rights in this invention.FIELD OF THE INVENTION [0003] The invention provides methods and compositions for identifying and characterizing functionally related gene products that are associated with mRNA-protein (mRNP) complexes and for characterizing cellular gene expression. The invention also provides methods and compositions for identifying and characterizing therapeutic targets and therapeutics. BACK...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70C12Q1/68G01N33/53C07H21/02G01N33/68
CPCC07H21/02C12Q1/68G01N33/6848G01N33/6845G01N33/68C12Q1/6804C12Q1/6809C12Q2600/158G01N33/5008G01N33/5014G01N33/502G01N33/5308C12Q2522/101
Inventor KEENE, JACKTENENBAUM, SCOTTCARSON, CRAIGPHELPS, WILLIAM
Owner RIBONOMICS
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