Cell based methods and systems for the identification of RNA regulatory sequences and compounds that modulate their functions
The present invention is directed to methods of screening for a small molecule that modulates the ability of a RNA regulatory sequence to inhibit mRNA translation in a mammalian cell comprising incubating the mammalian cell expressing a reporter mRNA and the RNA regulatory sequence in the presence of the small molecule, wherein the RNA regulatory sequence is not attached to the reporter mRNA; and monitoring a reporter protein signal produced by the reporter mRNA, wherein a change in the signal of the reporter protein in the presence of the small molecule compared to the reporter protein signal in the absence of the small molecule indicates the small molecule modulates the ability of the RNA regulatory sequence to inhibit mRNA translation.
1. A method of identifying a small molecule compound that inhibits the ability of an hepatitis C virus-internal ribosome entry site (HCV-IRES) mRNA regulatory sequence transcript fragment encoded by a sequence consisting of SEQ ID NO: 26 from inhibiting mRNA translation in a mammalian cell culture comprising
incubating the mammalian cell expressing a reporter mRNA transcript and the mRNA regulatory sequence transcript fragment in the presence of said small molecule compound, wherein said mRNA regulatory sequence transcript fragment is not attached to said reporter mRNA; and wherein said HCV-IRES mRNA regulatory sequence transcript fragment inhibits translation of the reporter mRNA transcript in said mammalian cell in the absence of said small molecule compound; and
monitoring a reporter protein signal produced by said reporter mRNA transcript in the presence of said small molecule compound, wherein an increase in the signal of the reporter protein in the presence of said small molecule compound compared to the reporter protein signal in the absence of said small molecule compound indicates said small molecule compound inhibits the ability of said mRNA regulatory sequence transcript fragment to inhibit translation of the reporter mRNA transcript.
2. The method of claim 1 , wherein said incubating step includes preincubating said reporter mRNA transcript expressed by the mammalian cell with the small molecule compound, and introducing said mRNA regulatory sequence transcript fragment into a preincubated system.
3. The method of claim 1 , wherein said reporter protein signal is fluorescence or bioluminescence.
4. The method of claim 1 , wherein said mammalian cell is selected from the group consisting of HeLa, HEL, MRC-5, NHFL, HCT 116, HEK 293, HEK 293T, Jurkat, human foreskin fibroblasts, A 549 and Caco-2.
5. The method of claim 1 , wherein said incubating step comprises incubating said small molecule compound with said mammalian cell expressing said reporter mRNA transcript followed by expression of said mRNA regulatory sequence transcript fragment in said mammalian cell.
6. The method of claim 1 , wherein said incubating step comprises expressing said reporter mRNA transcript and said mRNA regulatory sequence transcript fragment in said mammalian cell followed by introduction of said small molecule compound in said mammalian cell.
7. A method of identifying a small molecule compound that inhibits an hepatitis C virus-internal ribosome entry site (HCV-IRES) mRNA regulatory sequence transcript fragment encoded by a sequence selected from the group consisting of—SEQ ID NO 20, SEQ ID NO 32, SEQ ID NO 38, SEQ ID NO 41, and SEQ ID NO 44 from inhibiting mRNA translation in a mammalian cell culture comprising
incubating the mammalian cell expressing a reporter mRNA transcript and the mRNA regulatory sequence transcript fragment in the presence of said small molecule compound, wherein said mRNA regulatory sequence transcript fragment is not attached to said reporter mRNA transcript; and wherein said HCV-IRES mRNA regulatory sequence transcript fragment inhibits translation of the reporter mRNA transcript in said mammalian cell in the absence of said molecule compound; and
monitoring a reporter protein signal produced by said reporter mRNA transcript in the presence of said small molecule compound, wherein an increase in the signal of the reporter protein in the presence of said small molecule compound compared to the reporter protein signal in the absence of said small molecule compound indicates said small molecule compound inhibits the ability of said mRNA regulatory sequence transcript fragment to inhibit translation of the reporter mRNA transcript.
8. The method of claim 1 , wherein said incubating step includes preincubating said mRNA regulatory sequence transcript fragment with said small molecule compound, and introducing said reporter mRNA transcript into a preincubated system.