Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-08-14
2001-11-06
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S193000, C435S252300, C435S254110, C435S325000, C435S320100, C435S348000, C435S349000, C435S350000, C435S351000, C435S352000, C435S353000, C435S354000, C435S358000, C435S363000, C435S366000, C435S006120, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06312926
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to RNA polymerases, and particularly to enzymes involved in adding the cap structure to mRNA transcripts. The present invention further relates to transcription and translation of mRNA.
BACKGROUND OF THE INVENTION
Eukaryotic mRNAs contain a 5′-terminal cap, m
7
GpppN (1). This important modification of RNA polymerase II (pol II) transcripts occurs soon after they attain a chain length of 25-30 nucleotides (2-4). At this stage in transcription the C-terminal domain (CTD) of the pol II largest subunit is hyperphosphorylated (5, 6), and capping enzyme (CE) then binds to it (7-9). In mammals, CE is a bifunctional protein consisting of N-terminal RNA 5′-triphosphatase and C-terminal guanylyltransferase domains (7, 10). The combined effect of these activities on nascent pre-mRNAs is the conversion of 5′-terminal pppN to GpppN. Subsequent N7-methylation of GpppN 5′ ends to form m
7
GpppN caps is catalyzed by RNA (guanine-7-) methyltransferase (1, 11, 12). The 7-methylguanosine (m
7
G) moiety of the cap is a key feature in several aspects of RNA metabolism including transcript stability (13, 14), processing (15-17), transport to the cytoplasm (18, 19) and initiation of translation (1, 20).
Several functions of the cap structure are mediated by a family of cap-binding protein complexes that specifically recognize m
7
GpppN (19, 21). For example, in the nucleus, a cap-binding protein complex facilitates pre-mRNA splicing accuracy and efficiency and possibly nuclear export (19). In the cytoplasm, the heterotrimeric initiation factor eIF4F, which includes the cap-binding subunit eIF4E, promotes ribosome binding and translation initiation (21, 22). Although capping stabilizes mRNAs (13, 14), cap m
7
G is also recognized by the yeast decapping enzyme (23), and loss of the blocked 5′ end leads to 5′→3′ exonucleolytic degradation (13, 24). Consistent with the multiple effects of cap on gene expression, the RNA (guanine-7-) methyltransferase, like the RNA 5′-triphosphatase (25) and guanylyltransferase (26), is essential for viability in yeast (27).
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid encoding a mammalian capping enzyme. The present invention further provides an isolated nucleic acid encoding a mammalian (Guanine-7-) methyltransferase enzyme.
The present invention also provides an isolated mammalian capping enzyme protein or subunit thereof. In addition the present invention provides an isolated mammalian (Guanine-7-) methyltransferase enzyme protein or portion thereof.
It is an object of the present invention to provide a method for catalyzing formation of RNA 5′-terminal GpppN cap complex.
It is a further object of the present invention to provide a method for coupled formation of RNA 5′-terminal GpppN cap complex and translation of the RNA 5′-terminal GpppN cap complex in a cell-free extract.
It is a still further object of the present invention to provide a method for coupling RNA transcription and catalyzed formation of RNA 5′-terminal GpppN cap complex and translation of the RNA 5′-terminal GpppN cap complex in a cell-free extract comprising.
It is also an object of the present invention to provide a method for catalyzing formation of a methylated RNA 5′-terminal GpppN cap complex.
It is an even further object of the present invention to provide a kit for catalyzing formation of a methylated RNA 5′-terminal GpppN cap complex.
It is also a further object of the present invention to provide a kit for producing protein from a DNA template through coupled transcription, catalyzed formation of RNA 5′-terminal GpppN cap complex and translation.
Even still further it is an object of the present invention to provide a kit for producing protein from an uncapped RNA template through coupled catalyzed formation of RNA 5′-terminal GpppN cap complex and translation.
It is yet another object of the present invention to provide a mammalian RNA polymerase II/capping enzyme composition comprising RNA polymerase II and the provided mammalian capping enzyme protein.
Finally, it is an object of the present invention to provide a method for inhibiting degradation of an RNA transcript comprising contacting the RNA transcript with the provided mammalian capping enzyme protein under conditions permissive to the formation of a complex between the RNA transcript and the mammalian capping enzyme protein.
The present invention also contemplates using the provided nucleic acids, plasmids vectors and cells in an complementation assays in order to identify, screen, correct, treat and/or monitor a genetic defect in the capping pathway.
REFERENCES:
Sigma Catalog (Jan. 1993) Catalog No. A2383, p. 48.*
Yue et al., (Nov. 1997) Mammalian capping enzyme complements mutant . . . . Proc. Natl. Acad. Sci. USA 94: 12898-12903.*
Yamada-Okaba et al. (Apr. 1998) Isolation and characterization of a human cDNA for . . . . Nucleic Acids Research 26 (7): 1700-1706.*
Tsukamoto et al. (Feb. 1998) Cloning and characterization of two human cDNAs encoding the mRNA capping enzyme. Biochemical Biophysical Research Communications 243 (1): 101-108.*
Ho et al. (Apr. 1998) The guanyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 273 (16): 9577-9585.
Maldonado Edio
Pillutla Renuka
Reinberg Danny
Shatkin Aaron J.
Yue Zhenyu
Hutson Richard
Klauber & Jackson
Prouty Rebecca E.
University of Medicine & Dentistry of New Jersey
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