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Assay for identifying compounds which affect stability of mrnaRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidAssay for identifying compounds which affect stability of mrna description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070190532, Assay for identifying compounds which affect stability of mrna. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF INVENTION [0001] The present invention relates to the field of biological assays and in particular to an assay for the identification of biologically active compounds which have an effect on RNA stability. BACKGROUND OF THE INVENTION [0002] Messenger RNA expression in mammalian cells is highly regulated. Traditionally, emphasis has been placed on elucidating mechanisms by which genes are regulated at the transcriptional level; however, steady-state levels of mRNA is also dependent on its half-life or degradation rate. Changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes and different mechanisms have been proposed for the regulation of RNA turnover (Cleveland and Yen, 1989, New Biol. 1:121; Mitchell and Tollervey, 2000, Curr. Opin. Genet. Dev. 10:193; Mitchell and Tollervey, 2001, Curr. Opin. Cell. Biol. 13:320; Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237). However, the regulation of mRNA stability is complex. Regulation can involve sequence elements in the mRNA itself, activation of nucleases, as well as the involvement of complex signal transduction pathway(s) that ultimately influence trans-acting factors' interaction with mRNA stability sequence determinants. [0003] Recently, it has become increasingly apparent that the regulation of RNA half-life plays a critical role in the tight control of gene expression and that mRNA degradation is a highly controlled process. RNA instability allows for rapid up- or down-regulation of mRNA transcript levels upon changes in transcription rates. A number of critical cellular factors, e.g. transcription factors such as c-myc, or gene products which are involved in the host immune response such as cytokines, are required to be present only transiently to perform their normal functions. Transient stabilisation of the mRNAs which code for these factors permits accumulation and translation of these messages to express the desired cellular factors when required; whereas, under nonstabilised, normal conditions the rapid turnover rates of these mRNAs effectively limit and "switch off` expression of the cellular factors. Thus, aberrant MRNA turnover usually leads to altered protein levels, which can dramatically modify cellular properties. [0004] The stabilization of mRNA appears to be a major regulatory mechanism involved in the expression of inflammatory cytokines, growth factors, and certain proto-oncogenes. In the diseased state, mRNA half-life and levels of disease-related factors are significantly increased due to mRNA stabilization (Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237). Transcription rates and mRNA stability are often tightly and coordinately regulated for transiently expressed genes such as c-myc and c-fos, and cytokines such as IL-1, IL-2, IL-3, TNF.alpha., and GM-CSF. In addition, abnormal regulation of mRNA stabilisation can lead to unwanted build up of cellular factors leading to undesirable cell transformation, e.g. tumour formation, or inappropriate and tissue damaging inflammatory responses. [0005] Although the mechanisms which control mRNA stability are far from understood, sequence regions have been identified in a number of mRNAs, which appear to confer instability on the mRNAs which contain them. These sequence regions are referred to herein as "mRNA instability sequences". For example, typical mRNA instability sequences are the AREs (adenylate/uridylate (AU) rich elements), which are found in the 3' UTR (3' untranslated region) of certain genes including a number of immediate early genes and genes coding for inflammatory cytokines, e.g. IL-1.beta. and TNF.alpha.. The best characterized AU-rich element is the so-called Shaw-Kamen box or AUUUA motif (Shaw and Kamen, 1986, Cell 46:659). Multiple AUUUA sequences (in close proximity or in tandem) or AU-rich regions have been implicated in mRNA instability. For example, mRNA instability sequences described in the literature references identified below contain one or more copies of sequence motifs, e.g. selected from: AUUUA; UAUUUAU; UUAUUUA(U/A)(U/A), and AUUUAUUUA. Typically, in order to function as an instability determinant, the AUUUA motifs should be arranged in tandem, forming at least one UUAUUUAU/AU/A element (Lagnado et al., 1994, Mol. Cell. Biol. 14:7984). [0006] The following publications include extensive discussion of mRNA instability sequences and AREs, the sequences motifs, which they contain and (minimum) sequence requirements for mRNA destabilisation, as well as identifying a number of mRNA instability sequences and the genes which contain them: [0007] Shaw and Kamen, Cell, 1986, 46:659-667 (GM-CSF); [0008] Shyu et al., Genes & Development, 1991, 15:221-231 (c-fos); [0009] Sachs, Cell, 1993, 74:413-421 (Review. "Messenger RNA Degradation in Eukaryotes"); [0010] Chen et al., Mol. Cell. Biol., 1994, 14:416-426 (c-fos); [0011] Akashi et al., Blood, 1994, 83:3182-3187 (GM-CSF etc.); [0012] Nanbu et al., Mol. Cell. Biol., 1994, 14:4920-4920 (uPA); [0013] Stoecklin et al., J. Biol. Chem., 1994, 269:28591-28597 (IL-3); [0014] Lagnado et al., Mol. Cell. Biol., 1994, 14:7984-7995 (general); [0015] Zhang et al., Mol. Cell. Biol., 1995, 15:2231-2244 (yeast); [0016] Zubiaga et al., Mol. Cell. Biol., 1995, 15:2219-2230 (general); [0017] Winstall et al., Mol. Cell. Biol., 1995, 15:3796-3804 (c-fos, GM-CSF); [0018] Chen et al., Mol. Cell. Biol., 1995, 15:5777-5788 (c-fos, GM-CSF); [0019] Chen et al., TIBS, 1995, 20:465-470 (review); [0020] Levy et al., J. Biol. Chem., 1996, 271:2746-2753 (VEGF); [0021] Kastelic et al., Cytokine, 1996, 8:751-761; [0022] Crawford et al., J. Biol. Chem., 1997, 272:21120-21127 (TNF.alpha.); [0023] Xu et al., Mol. Cell. Biol., 1997, 18:4611-4621 (general); [0024] Danner et al., J. Biol. Chem., 1998, 273:3223-3229 (human .beta.2-Adrenergic Receptor); [0025] Lewis et al., J. Biol. Chem., 1998, 273:13781-13786 (TNF.alpha.); [0026] Chen, C.-Y. and Shyu, A.-B., Mol. Cell. Biol., 1994, 14:8471-8482; and [0027] Klausner, R. et al., Cell, 1993, 72:19-28. [0028] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. SUMMARY OF THE INVENTION [0029] An object of the present invention is to provide an assay for identifying compounds which affect stability of mRNA. In accordance with an aspect of the present invention, there is provided a DNA expression vector comprising: a first DNA sequence comprising the coding sequence for one or more protein having a detectable signal; one or more 3' UTR sequence and one or more expression control sequence operatively associated with said coding sequence, and a heterologous instability sequence DNA inserted into said 3' UTR sequence comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes. [0030] In accordance with another aspect of the invention, there is provided a stably transfected cell line comprising: a DNA expression vector comprising a first DNA sequence encoding a first protein having a detectable signal, one or more 3' UTR sequence and one or more expression control sequence operatively associated with said first DNA sequence, and a heterologous instability sequence DNA inserted into said 3' UTR sequences, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes; and a control DNA expression vector comprising a control DNA sequence encoding a second protein having a detectable signal, and one or more 3' UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence. [0031] In accordance with another aspect of the invention, there is provided a method of screening for one or more compound which affect mRNA stability comprising the steps of: [0032] i) providing a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence; [0033] ii) contacting said DNA expression vector with at least one test compound under conditions whereby, in the absence of the test compound, said DNA expression system is capable of expressing said protein having a detectable signal; [0034] iii) measuring said detectable signal; and [0035] iv) comparing the measured detectable signal with a control, wherein a decrease in the measured detectable signal compared to said control indicates a compound that decreases mRNA stability and an increase in the measured detectable signal compared to said control indicates a compound that increases mRNA stability. [0036] In accordance with another aspect of the invention, there is provided a method for comparing the extent of mRNA degradation induced by two or more compounds comprising the steps of: [0037] i) providing a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence; [0038] ii) contacting said DNA expression vector separately with two or more test compounds under conditions whereby, in the absence of the test compounds, said DNA expression system is capable of expressing said protein having a detectable signal; [0039] iii) measuring said detectable signal in the presence of each test compound; and [0040] iv) comparing the measured detectable signals; wherein a lower measured detectable signal indicates a greater extent of mRNA degradation. [0041] In accordance with another aspect of the invention, there is provided an assay system for screening for compounds which destabilise mRNA comprising: [0042] i) a DNA expression vector comprising a first DNA sequence encoding a first protein having a detectable signal, one or more 3' UTR sequence and one or more expression control sequence operatively associated with said DNA sequence, and a heterologous instability sequence DNA inserted into said 3' UTR sequences, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes; and [0043] ii) a control DNA expression vector comprising a control DNA sequence encoding a control protein having a detectable signal, and one or more 3' UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence. [0044] In accordance with another aspect of the invention, there is provided a high throughput method for screening libraries of compounds to identify compounds that affect the stability of mRNA comprising: [0045] i) providing a stably transfected cell line comprising a DNA expression vector, which in the absence of a test compound is capable of expressing a protein having a detectable signal, wherein the mRNA which is transcribed from said expression vector and encodes said protein comprises at least one copy of a heterologous mRNA instability sequence; [0046] ii) inoculating wells of one or more multi-well plates comprising a growth medium with said cell line; [0047] iii) maintaining said one or more multi-well plates under conditions that allow cells of said cell line to grow and express said protein having a detectable signal; [0048] iv) contacting the cells with one or more test compound; [0049] v) measuring said detectable signal; and [0050] vi) comparing the measured detectable signal with a control; wherein a decrease in the measured detectable signal compared to said control indicates a compound that decreases mRNA stability and an increase in the measured detectable signal compared to said control indicates a compound that increases mRNA stability. [0051] In accordance with another aspect of the invention, there is provided a kit comprising an assay system for screening for compounds which destabilize mRNA, said assay system comprising: [0052] i) one or more DNA expression vector comprising a first DNA sequence encoding a protein having a detectable signal, one or more 3' UTR sequence and one or more expression control sequence operatively associated with said first DNA sequence, and a heterologous instability sequence DNA inserted into said 3' UTR sequences, said instability sequence DNA comprising a second DNA sequence corresponding to one or more mRNA instability sequence derived from one or more naturally occurring genes; and [0053] ii) a control DNA expression vector comprising a control DNA sequence encoding a second protein having a detectable signal, one or more 3' UTR sequence and one or more expression control sequence operatively associated with said control DNA sequence; and optionally [0054] iii) instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS [0055] FIG. 1 shows the DNA sequence of IL-1.beta. 3'UTR; [0056] FIG. 2 shows the 30 bp fragment used as a mRNA instability sequence in Example 1; [0057] FIG. 3A shows plasmid diagrams for pGL2_Neo30 and pGL2-Control; [0058] FIG. 3B shows plasmid diagram for pGL2-.beta.-galactosidase; [0059] FIG. 4 shows graphs of luciferase activity over the time of differentiation for clone No. 53 (A) and clone No. 63 (B); [0060] FIG. 5 shows graphs of luciferase half lives, 4 and 8 hours after addition of compounds for clones No. 53 and 63 treated with radicicol analog A (RAA), actinomycin D (act D.) and cyclohexamide (CHX); Continue reading about Assay for identifying compounds which affect stability of mrna... 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