Compositions and methods for reproducing and modulating mammalian messenger RNA decapping
View Patent ↗An in-vitro system in which mammalian messenger RNA decapping occurs is provided, for use in identifying modulators, deficiencies, and other aspects of the regulation of RNA turnover.
1. A mammalian in vitro mRNA decapping system comprising:
a) polysome-free HeLa cell cytoplasmic extract;
b) a methylated cap analog and
c) a cap-labeled mRNA substrate.
2. The mammalian in vitro mRNA decapping system of claim 1 wherein said methylated cap analog is 7me GpppG or 7me GTP.
3. The mammalian in vitro mRNA decapping system of claim 1 wherein said cap-labeled mRNA substrate is labeled at the alpha phosphate of the cap.
4. The mammalian in vitro mRNA decapping system of claim 1 wherein said cap-labeled mRNA substrate is labeled at the cap of said cap-labled mRNA substrate by a label selected from the group consisting of a radioactive label, a non-radioactive isotopic label, a fluorescent moiety, a visibly-detectable moiety, releasable substrate, a co-factor for a chemical reaction, and a co-factor for a enzymatic reaction.
5. The mammalian in vitro mRNA decapping system of claim 1 further comprising means for sequestering proteins that bind to poly(A).
6. The mammalian in vitro mRNA decapping system of claim 1 wherein said HeLa cell cytoplasmic extract is a HeLa S100 cell cytoplasmic extract.
7. The mammalian in vitro mRNA decapping system of claim 6 wherein said HeLa cell cytoplasmic extract is prepared by dialysis of said extract containing 10% glycerol.
8. The mammalian in vitro mRNA decapping system of claim 6 wherein said S100 cell cytoplasmic extract comprises a 100,000×g, 1 hour supernatant from a HeLa cell lysate.
9. The mammalian in vitro mRNA decapping system of claim 1 wherein said cap-labeled mRNA substrate comprises poly(A) or at lea one RNA element.
10. The manunalian in vitro mRNA decapping system of claim 9 wherein said RNA element is a pyrimidine-rich element.
11. The mammalian in vitro mRNA decapping system of claim 9 further comprising means for stimulating decapping of the cap-labeled mRNA substrate wherein the cap-labeled mRNA substrate comprises poly(A).
12. The mammalian in vitro mRNA decapping syst of claim 9 further comprising a cold poly(A) competitor RNA.
13. The mammalian in vitro mRNA decapping system of claim 9 wherein said RNA element is an AU-rich element.
14. The mammalian in vitro mRNA decapping system of claim 13 further comprising means for reducing decapping of the cap-labeled mRNA substrate.
15. The mammalian in vitro mRNA decapping system of claim 13 further comprising an AU-rich element competitor RNA.
16. A kit for measuring mRNA decapping in vitro comprising:
a) a polysome-free HeLa cell cytoplasmic extract;
b) a methylated cap analog; and
c) cap-labeled mRNA substrate.
17. The kit of claim 16 wherein said cap-labeled mRNA substrate is labeled at the alpha phosphate of the cap.
18. The kit of claim 16 wherein said cap-labeled mRNA substrate is labeled at the cap of said cap-labeled mRNA substrate by a label selected from the group consisting of a radioactive label, a non-radioactive isotopic label, a fluorescent moiety, a visibly-detectable moiety, a releasable substrate, a co-factor for a chemical reaction, and a co-factor for an enzymatic reaction.
19. The kit of claim 16 wherein the polysome-free HeLa cell cytoplasmic extract is HeLa S100 cell cytoplasmic extract.
20. The kit of claim 16 wherein the cap-labeled mRNA substrate comprises poly(A).
21. The kit of claim 20 further comprising means for stimulating decapping the cap labeled mRNA substrate.
22. The kit of claim 20 further comprising a cold poly(A) competitor RNA.
23. The kit of claim 16 wherein the cap-labeled nRNA substrate comprises an RNA element.
24. The kit of claim 23 wherein the RNA element is an AU-rich element.
25. The kit of claim 24 further comprising means for reducing decapping the cap-labeled mRNA substrate.
26. The kit of claim 24 further comprising an AU-rich element competitor RNA.
27. A mammalian in vitro mRNA decapping system comprising:
a) a polysome-free HeLa cell cytoplasmic extract;
b) a cap-labeled mRNA substrate; and
c) means for decapping the cap-labeled mRNA substrate.
28. The kit of claim 27 wherein the polysome-free HeLa cell cytoplasmic extract is a HeLa S100 cell cytoplasmic extract.
29. A kit for measuring mRNA decapping in vitro comprising:
a) a polysome-free HeLa cell cytoplasmic extract;
b) a cap-labeled mRNA substrate; and
c) means for decapping the cap-labeled mRNA substrate.
30. The kit of claim 29 wherein the polysome-free HeLa cell cytoplasmic extract is a HeLa S100 cytoplasmic extract.