Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-26
2003-09-30
Moran, Marjorie (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007210, C435S367000, C435S372000, C435S455000, C536S025410
Reexamination Certificate
active
06627398
ABSTRACT:
FIELD OF THE INVENTION
Broadly, the present invention involves a system and method for monitoring the stability of RNA and identifying agents capable of modulating RNA stability.
BACKGROUND OF THE INVENTION
The relative stability of a mRNA is an important regulator of gene expression. The half-life of a mRNA plays a role in determining both the steady state level of expression as well as the rate of inducibility of a gene product. In general, many short-lived proteins are encoded by short-lived mRNAs. Several mRNAs that encode stable proteins, such as &agr;-globin, have also been shown to have extraordinarily long half-lives. Surveillance mechanisms are also used by the cell to identify and shorten the half-lives of mRNAs that contain nonsense codon mutations. Clearly, changes in the half-life of a mRNA can have dramatic consequences on cellular responses and function.
Little is known about mechanisms of mRNA turnover and stability in mammalian cells, but in vivo data are beginning to allow some generalizations about major pathways of mRNA turnover. The mRNA poly(A) tail can be progressively shortened throughout the lifetime of a mRNA in the cytoplasm. Controlling the rate of this deadenylation process appears to be a target for many factors that regulate mRNA stability. Once the poly(A) tail is shortened to approximately 50-100 bases, the body of the mRNA is degraded in a rapid fashion with no discernible intermediates. The process of translation also influences mRNA stability. Little is known, however, concerning the enzymes and regulatory components involved in mammalian mRNA turnover.
Several cis-acting elements have been shown to play a role in mRNA stability. Terminal (5′) cap and 3′-poly(A) structures and associated proteins are likely to protect the transcript from exonucleases. Several destabilizing as well as stabilizing elements located in the body of the mRNA have also been identified. The best characterized instability element is an A-U rich sequence (ARE) found in the 3′ untranslated region of many short-lived mRNAs. These AREs primarily consist of AUUUA (SEQ ID NO: 12) repeats or a related nonameric sequence. AREs have been shown to increase the rate of deadenylation and mRNA turnover in a translation-independent fashion. For example, proteins with AU-rich elements include many growth factor and cytokine mRNAs, such as c-fos, c-jun, c-myc TNF&agr;, GMCSF, IL1-15, and IFN-&bgr;. Other stability elements include C-rich stabilizing elements, such as are found in the mRNAs of globin, collagen, lipoxygenase, and tyrosine hydroxylase. Still other mRNAs have as yet uncharacterized or poorly characterized sequence elements, for example, that have been identified by deletion analysis, e.g. VEGF mRNA.
Numerous proteins have been described that interact with some specificity with an ARE, bat their exact role in the process of mRNA turnover remains to be defined. For example, proteins which bind to the ARE described above include HuR and other ELAv family proteins, such as HuR (also called HuA), Hel-N1 (also called HuB), HuC and HuD; AUF 1 (four isoforms); tristetraprolin; AUH; TIA; TIAR; glyceraldebyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. Many others have not been extensively characterized
Through the application of genetics, the mechanisms and factors involved in the turnover of mRNA in
Saccharomyces cerevisiae
are beginning to be identified. One major pathway of mRNA decay involves decapping followed by the action of a 5′-to-3′ exonuclease. Evidence has also been obtained for a role for 3′-to-5′ exonucleases in an alternative pathway. Functionally significant interactions between the cap structure and the 3′ poly(A) tail of yeast mRNAs have also been described. Several factors involved in the translation-dependent pathway of nonsense-codon-mediated decay have also been identified. Whether these observations are generally applicable to mammalian cells, however, remains to be established.
Mechanistic questions in mammalian cells are usually best approached using biochemical systems due to the inherent difficulties with mammalian cells as a genetic system. Thus, efforts have been made to develop in vitro systems to study mRNA stability and turnover. However, the presently available in vitro systems suffer from numerous limitations. For example, many suffer from poor data quality and a general lack of reproducibility that significantly limits their application. Another key problem is that most of these systems do not faithfully reproduce all aspects of mRNA stability. A significant difficulty in the development of these systems is to differentiate between random, non-specific RNA degradation and true, regulated mRNA turnover. The significance of all previous in vitro systems to the true in vivo process of mRNA stability, therefore, is unclear. To date, no in vitro mRNA stability system has been generally accepted in the field as valid and useful. Other problems that have been uncovered in presently available systems are that they usually involve a complicated extract protocol that is not generally reproducible by other laboratories in the field. Also, presently available systems can only be used to assess the stability of endogenous mRNAs, severely limiting their utility. Finally, the data quality obtained using such systems is highly variable, precluding their use in sensitive screening assays.
Accordingly, there exists a need for an in vitro RNA stability system is efficient and highly reproducible, and further, one which produces minimal to undetectable amounts of RNA degradation
A further need exists for an in vitro RNA stability system wherein deadenylation of an RNA transcript in the system should occur before general degradation of the mRNA body is observed. Also needed is an in vitro RNA stability system wherein degradation of the mRNA body occurs in an apparently highly processive fashion without detectable intermediates, and further, the regulation of the rate of overall deadenylation and degradation should be observed in a sequence-specific manner. Such a system should be applicable to exogenous RNAs and allow ease of experimental manipulation.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
In accordance with the present invention, an in vitro system for modulating the stability and turnover of an RNA molecule is provided which models RNA processing in vivo. Thus, the present invention permits high throughput screening of compounds/macromolecules that modulate the stability of eukaryotic RNAs in order to identify and design drugs to affect the expression of selected transcripts, as well as to aid in the characterization of endogenous proteins and other macromolecules involved in mRNA stability. The in vitro system of the present invention is useful as a diagnostic aid for determining the molecular defect in selective disease alleles; development of in vitro mRNA stability systems for other eukaryotic organisms including parasites and fungi which should lead to novel drug discovery; and improving gene delivery systems by using the system to identify factors and RNA sequences that affect RNA stability.
Broadly, the present invention extends to an in vitro system capable of recapitulating regulated RNA turnover of an exogenously added preselected target RNA sequence, the system comprising a cell extract and a target RNA sequence. In a non-limiting example of the system described herein, the regulated RNA turnover is AU-rich element regulated RNA turnover or C-rich element regulated RNA turnover.
The cell extract of the system of the present invention is isolated from lysed eukaryotic cells or tissues; the cell extract may be obtained for example from a cell line, such as HeLa cells or a T cell line, but the invention is not so limited. The cell extract may be prepared from cells comprising foreign nucleic acid, such as those that are infected, stably transfected, or transiently
Ford Lance P.
Wilusz Jeffrey
Moran Marjorie
Perkins Coie LLP
University of Medicine and Dentistry of New Jersey
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