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Methods for isolating and characterizing endogenous mrna-protein (mrnp) complexes

Methods for isolating and characterizing endogenous mrna-protein (mrnp) complexes description/claims

This application is a continuation of U.S. patent application Ser. No. 10/238,306, filed on Sep. 10, 2002, which is a continuation of U.S. patent application Ser. No. 09/750,401, filed on Dec. 28, 2000, now issued as U.S. Pat. No. 6,635,422, which claims the benefit of U.S. Provisional Application No. 60/173,338, filed Dec. 28, 1999, the contents of which are hereby incorporated in their entirety.

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 to this invention.

This invention relates generally to post-transcriptional regulation and methods of profiling gene expression.

Many diseases are genetically based, and the genetic background of each individual can have a profound effect on his or her susceptibility to disease. The relatively new field of functional genomics has provided researchers with the ability to determine the functions of proteins based upon knowledge of the genes that encode the proteins. A major goal of functional genomics is to identify gene products that are suitable targets for drug discovery. Such knowledge can lead to a basis for target validation if it is demonstrated that the target of interest has an essential function in a disease. Accordingly, a need exists to develop methods that allow profiling of the gene expression state of cells and tissues in order to understand the consequences of genetics on growth and development.

Understanding global gene expression at the level of the whole cell requires detailed knowledge of the contributions of transcription, pre-mRNA processing, mRNA turnover and translation. Although the sum total of these regulatory processes in each cell accounts for its unique expression profile, few methods are available to independently assess each process en masse.

The expression state of genes in a complex tissue or tumor is generally determined by extracting messenger RNAs from samples (e.g., whole tissues) and analyzing the expressed genes using cDNA libraries, microarrays or serial analysis of gene expression (SAGE) methodologies. See, e.g., Duggan, et al., (1999) Nature Genetics 21, 10-14; Gerhold, et al., (1999) Trends in Biochemical Sciences 24, 168-173; Brown, et al, (1999) Nature Genetics 21, 38-41; Velculescu, et al., (1995) Science 270, 484-487 Velculescu, et al. (1997) Cell 88, 243-251. In order to determine the gene expression profile of any single cell type within a tissue or tumor or to recover those messenger RNAs, the tissue must first be subjected to microdissection. This is very laborious, as only a small amount of cellular material is recovered and the purity as well as the quality of the cellular material is compromised.

Post-transcriptional events influence the outcome of protein expression as significantly as transcriptional events. The regulation of transcription and post-transcription are generally linked. Altering the expression of transcriptional activators or repressors has important consequences for the development of a cell. Therefore, feedback loops following translational activation of specific mRNAs may change the program of transcription in response to growth or differentiation signals. DNA arrays are well-suited for profiling the steady-state levels of mRNA globally (i.e., total mRNA or the “transcriptome”). However, because of post-transcriptional events affecting mRNA stability and translation, the expression levels of many cellular proteins do not directly correlate with steady-state levels of mRNAs (Gygi et al. (1999) Mol. Cell. Biol. 19, 1720-1730; Futcher et al. (1999) Mol. Cell. Biol. 19, 7357-7368).

Many mRNAs contain sequences that regulate their post-transcriptional expression and localization (Richter (1996) in Translational Control, eds. J. W. B Hershey, et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 481-504). These regulatory elements reside in both introns and exons of pre-mRNAs, as well as in both coding and noncoding regions of mature transcripts (Jacobson and Peltz (1996) Annu. Rev. Biochem. 65, 693-739; Wickens et al. (1997) Curr. Opin. Genet. Dev. 7, 220-232). One example of a sequence-specific regulatory motif is the AU-rich instability element (ARE) present in the 3′-untranslated regions (UTRs) of early-response gene (ERG) mRNAs, many of which encode proteins essential for growth and differentiation (Caput et al. (1986) Proc. Natl. Acad. Sci. USA 83, 1670-1674; Shaw and Kamen (1986) Cell 46, 659-667; Schiavi et al. (1992) Biochim. Biophys. Acta 1114, 95-106; Chen and Shyu (1995) Trends Biochem. Sci. 20, 465-470). Regulation via the ARE is poorly understood, but the mammalian ELAV/Hu proteins have been shown to bind to ARE sequence elements in vitro and to affect post-transcriptional mRNA stability and translation in vivo (Jain et al (1997) Mol. Cell. Biol. 17, 954-962; Levy et al. (1998) J. Biol. Chem. 273, 6417-6423; Fan and Steitz (1998) EMBO J. 17, 3448-3460; Peng et al. (1998) EMBO J. 17, 3461-3470; Keene (1999) Proc. Natl. Acad. Sci. USA 96, 5-7).

In vitro RNA selection methods based upon cellular sequences are reported in Gao et al., Proc. Natl. Acad. Sci. USA 90, 11207-11211 (1994) and U.S. Pat. Nos. 5,773,246, 5,525,495 and 5,444,149, all to Keene et al., the disclosures of which are incorporated herein in their entirety. Generally, these methods were intended to identify large numbers of mRNAs present in messenger RNP (mRNP) complexes, and utilized in vitro binding and amplification of mRNA sequences from large pools of naturally-occurring mRNAs. These studies used proteins (referred to as ELAV or Hu proteins) known to bind to AU-rich sequence elements present in the untranslated regions of cellular mRNAs. These experiments led to the discovery that mRNAs which are structurally or functionally related may be revealed using multi-targeted RNA binding proteins (i.e., RNA binding proteins that specifically bind more than one target). See Levine, et al, (1994) et al., Molecular and Cellular Biology 13, 3494-3504; and King, et al., (1993) Journal of Neuroscience 14, 1943-1952; reviewed in Antic and Keene (1997) American Journal of Human Genetics 61, 273-278 and Keene (1999) Proceedings of the National Academy of Sciences (USA) 96, 5-7. However, these reports are limited to in vitro applications, and do not describe in vivo methods for partitioning RNA into structural or functional subsets using RNA binding proteins. Although in vitro methods have been used to determine protein-RNA interactions, their use has certain limitations. Biochemical methods are generally reliable when carefully controlled, but RNA-binding can be problematic because many interactions may be of low affinity, low specificity or even artifactual. In order to understand RNA-protein interactions and their functional implications on a global systems level it is necessary to find reliable methods to monitor messenger RNP complexes in vivo.

The successful immunoprecipitation of epitope-tagged ELAV/Hu protein which has been transfected into pre-neuronal cells has been reported. See Antic et al., Genes and Development 13, 449-461 (1999). This immunoprecipitation was followed by nucleic acid amplification that allowed for the identification of a messenger RNA encoding neurofilament M protein (NF-M).

The present invention relates to a new, in vivo approach for the determination of gene expression that utilizes the flow of genetic information through messenger RNA clusters or subsets. Recently, the practice of examining multiple macromolecular events simultaneously and in parallel with the goal of organizing such information computationally has taken the designation “-ome.” Thus, the genome identifies all of the genes of a cell, while the transcriptome is defined as the messenger RNA complement of the genome and the proteome is defined as the protein complement of the genome (see FIG. 1). The present inventors have defined several physically organized subsets of the transcriptome and defined them as dynamic units of the “ribonome”. As described herein, the ribonome consists of a plurality of distinct subsets of messenger RNAs (mRNAs) that are clustered in the cell due to their association with RNA-binding proteins (e.g., regulatory RNA-binding proteins). By identifying the mRNA components of a cellular ribonome, the cellular transcriptome can be broken down into a series of subprofiles that together can be used to define the gene expression state of a cell or tissue (see FIG. 2). In combination with, for example, high throughput approaches and by multiplexing RNA processing assays, the present inventive methods provide the ability to determine the changes that occur in multiple gene transcripts simultaneously.

Accordingly, one aspect of the invention is an in vivo method of partitioning endogenous, cellular mRNA-binding protein (mRNP) complexes. The method, in one embodiment, comprises contacting a biological sample that comprises at least one mRNP complex with a ligand that specifically binds a component of the mRNP complex. The biological sample may be, for example, a tissue sample, whole tissue, a whole organ, a cell culture, or a cell extract or lysate. The component of the mRNP complex may be a RNA binding protein, a RNA-associated protein, a nucleic acid associated with the mRNP complex including the mRNA itself, or another molecule or compound (e.g., carbohydrate, lipid, vitamin, etc.) that associates with the mRNP complex. The ligand may be, for example, an antibody that specifically binds the component, a nucleic acid that binds the component (e.g., an antisense molecule, a RNA molecule that binds the component), or any other compound or molecule that binds the component of the complex. The mRNP complex is then separated by binding the ligand (now bound to the mRNP complex) to a binding molecule that binds the ligand. The binding molecule may bind the ligand directly (i.e., may be an antibody specific for the ligand), or may bind the ligand indirectly (i.e., may be an antibody or binding partner for a tag on the ligand). The binding molecule will be attached to a solid support, such as a bead or plate or column, as known in the art. Accordingly, the mRNP complex will be attached to the solid support via the ligand and binding molecule. The mRNP complex is then collected by removing it from the solid support (i.e., the complex is washed off the solid support using suitable conditions and solvents).

The identity of the mRNA bound within the mRNP complex may then be determined, for example, by separating the mRNA from the complex, reverse transcribing the mRNA into cDNA, and sequencing the cDNA.

In embodiments of the invention, therefore, the mRNP complex may be isolated by direct immunoprecipitation of the mRNP complexes, either with or without epitope tags, or by other biochemical partitioning methods. For example, other proteins bound to or associated with the mRNP complex may be immunoprecipitated in order to recover the mRNP complex and subsequently the mRNAs bound within the complex. The skilled artisan will appreciate that embodiments of the inventive method allow for the identification of a plurality of mRNA complexes simultaneously (i.e., concurrently), sequentially, or in batch-wise fashion. Alternatively, the method may be carried out on one biological sample (or portion thereof) numerous times, the steps of the method being performed in a sequential fashion, with each iteration of the method utilizing a different ligand. In any of the described embodiments, cDNA or genomic microarray grids, for example, may be used to identify mRNAs isolated by the inventive method en masse.

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