Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-27
2001-10-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C530S350000
Reexamination Certificate
active
06303311
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
The biological function of nucleic acid binding proteins is often separable from their sequence specificity. In some cases, as with many RNA and DNA polymerases, a protein possessing the enzymatic activity site is directed to the correct location through its interaction with a distinct polypeptide. In others, as with many transcription factors and regulators of mRNA metabolism, function and sequence specificity reside in distinct domains of the same polypeptide. Dissection of the two domains from one another facilitates their detailed analysis, liberating each from the constraints of the other.
Poly(A) is a virtually universal feature of mRNAs in eukaryotes, and exists in the cytoplasm as an RNA-protein complex with Poly(A) binding protein (Pab1p). Like the tail, Pab1p is highly conserved. The presence of a poly(A) tail typically stabilizes mRNAs and can enhance their translation both in vivo and in vitro. It is unclear whether these biological functions reside solely in the protein, or require the RNA-protein complex.
The role of the Pab1p in stability has been demonstrated in the budding yeast Saccharomyces cerevesiae. The yeast PAB1 gene is required for viability (Sachs, 1986). The lethality of a pab1 deletion can be suppressed by mutations within various genes associated with mRNA turnover, such as xrn1 and dcpl, indicating Pab1p likely has an essential function in mRNA decay (Hatfield, 1996). Degradation of most yeast mRNAs is initiated by shortening of the 3′ poly(A) tail, followed by Dcplp cleavage of the 5′m
7
GpppG cap (“decapping”), which permits 5′ to 3′ exonucleolytic digestion by the XRN1 gene product (Decker, 1993; Muhlrad, 1994; Beelman, 1996). Decapping occurs only after the poly(A) tail has been shortened to 10-12 adenosine residues (Muhlrad, 1994), which corresponds provocatively with the minimal Pab1p binding site of 12 adenosines and suggests that eviction of the last Pab1p/poly(A) complex triggers turnover. Furthermore, in the absence of Pab1p, decapping becomes independent of poly(A) length (Caponigro, 1995).
The molecular link between poly(A) and the cap may be a tripartite protein bridge between the two ends of the mRNA. Pab1p, when bound to the poly(A) tail, interacts in vitro with two yeast proteins, TIF4631 and TIF4632, related to the translation initiation factor eIF-4G (Tarun, 1996). eIF-4G in turn interacts with eIF-4F, containing the cytoplasmic cap-binding protein, eIF-4E, an initiation factor itself. The in vitro association of Pab1p and eIF-4G is dependent on the poly(A) tail, and is consistent with earlier work demonstrating biochemical interactions between poly(A) and complexes containing eIF-4F in vitro (Gallie, 1994). The same tripartite bridge could explain poly(A) 's stimulation of translation in vitro and in vivo.
The interaction between Pab1p and eIF-4G, demonstrated in vitro, requires the presence of RNA, implying that the poly(A)/Pab1p complex likely is essential. Similarly, deadenylation in crude yeast extracts is dependent on the presence of Pab1p, implying that the yeast deadenylase may recognize not poly(A) alone, but the poly(A)/Pab1p complex (Lowell, 1992).
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method for determining the function of a test protein. In a preferred form of this method, one first fuses a test protein to an mRNA binding protein, preferably by first creating a chimeric gene sequence encoding both proteins in tandem. One then exposes the fusion protein to a recombinant reporter mRNA molecule under conditions suitable for the binding of the fusion protein to the reporter mRNA. The mRNA molecule comprises a binding site for the RNA-binding protein in the 3′ untranslated region. Once the fusion protein is bound (or “tethered”) to the reporter mRNA, one observes the properties of the reporter mRNA and correlates these properties with test protein function.
In a preferred form of the present invention, the RNA-binding protein is MS2 coat protein and the RNA-binding site is MS2 binding site.
In another preferred form of the present invention, the tethering takes place in yeast.
In a preferred form of the invention, a candidate inhibitor or enhancer compound is evaluated by exposing the compound to the tethered molecules.
Another form of the present invention is a tethered structure comprised of the reporter mRNA and fusion protein described above.
The present invention is also the tethered structure formed by binding between the fusion protein and the reporter mRNA.
It is an object of the present invention to analyze the function of a test protein by tethering the test protein to a reporter mRNA.
It is another object of the present invention to analyze candidate inhibitors and enhancers.
It is an advantage of the present invention that one need not know the natural RNA-binding sites of the test protein to analyze the protein's function.
Other objects, advantages and functions of the present invention will become apparent after one has reviewed the specification, claims and drawings herein.
REFERENCES:
E. Paraskeva, et al., “A Translational Repression Assay Procedure (TRAP) for RNA-protein Interactions In Vivo,”Proc. Natl. Acad. Sci. USA95(3) :951-956, 1998.
D.J. Sengupta, et al., “A Three-Hybrid System to Detect RNA-Protein Interactions In Vivo,”Proc. Nat. Acad. Sci. USA93:8496-8501, 1996.
R. Stripecke, et al., “Proteins Binding to 5′ Untranslated Region Sites: A General Mechanism for Translational Regulation of mRNAs in Human and Yeast Cells,”Mole. Cell. Biol.14(9) :5898-5909, 1994.
J. Valcárcel, et al., “The Protein Sex-Lethal Antagonizes the Splicing Factor U2AF to Regulate Alternative Splicing ofTransformerPre-mRNA,”Nature362:171-175, 1993.
M. Wickens, et al., “Translational Control of Developmental Decisions,”Translational Control, Cold Spring Harbor Laboratory Press, pp. 411-450, 1996.
M. Wickens, et al., “Life and Death in the Cytoplasm: Messages from the 3′ End,”Curr. Opin. Genet. & Develop.7:220-232, 1997.
Parker Roy
Wickens Marvin P.
Monshipouri M.
Prouty Rebecca E.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
LandOfFree
Tethered function assay for protein function does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Tethered function assay for protein function, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Tethered function assay for protein function will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2557217