Inhibition: RNA Aptamers
RNA Aptamers: A single strand of RNA folds back upon itself to give a complicated secondary structure. In three dimensions, this aptamer forms a shape which can bind tightly to its protein target.
To disrupt not only a specific protein, but a specific interacting domain on that protein, we turn to RNA aptamer technology. We select RNA aptamers from large, highly complex RNA pools using cycles of selection and amplification (SELEX). Aptamers provide several advantages as inhibitors:
- Like an antibody, they can be made to order specifically for a particular protein
- Like a small organic molecule, they can rapidly target a specific protein domain within cells
- Like a conditional allele, they are able to exert their effect in whole organisms, but
- They are also targetable to specific tissues, cells, or stages of development.
Tome JM, Ozer A, Pagano JM, Gheba D, Schroth GP, Lis JT. Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity
- Salamanca HH, Fuda N, Shi H, Lis JT. (2011) “An RNA aptamer perturbs heat shock transcription factor activity in Drosophila melanogaster.” Nucleic Acids Res. May 16. [Epub ahead of print]. (PubMed)
- Sevilimedu A, Shi H, and Lis JT (2008) “TFIIB aptamers inhibit transcription by perturbing PIC formation at distinct stages.”Nucleic Acids Res. 36:3118-27.
- Shi H et al. (2007) “RNA aptamers directed to discrete functional sites on a single protein structural domain.” Proc Natl Acad Sci U S A. 104(10):3742-6.(PubMed)
- Zhao X et al. (2006) “An RNA aptamer that interferes with the DNA binding of the HSF transcription activator.” Nucleic Acids Res. 34(13):3755-61. (PubMed)
- Fan X et al. (2005) “Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP.” Nucleic Acids Res. 33(3):838-45. (PubMed)
- Fan X et al. (2004) “Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes.” Proc Natl Acad Sci U S A. 101(18):6934-9. (PubMed)
- Shi H et al. (1999) “RNA aptamers as effective protein antagonists in a multicellular organism.” Proc Natl Acad Sci U S A. 96(18):10033-8. (PubMed)
- First demonstration that aptamers can interfere with a protein’s function in a multicellular organism.
RNAi can be used with relative ease in Drosophila cell cultures to deplete levels of specific proteins to less than 20% of their normal levels. By reducing the levels of a transcription, elongation, or processing factor, we can examine its role in various transcriptional and co-transcriptional processes. Generally we study the effect on the localization of other protein factors by ChIP, or on the transcripts produced by quantitative reverse transcription PCR or other methods.
- Fuda NJ, Buckley MS, Wei W, Core LJ, Waters CT, Reinberg D, Lis JT. (2012) “Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes.” Mol Cell Biol. 32(17):3428-37. (PubMed)
- Petesch SJ and Lis JT (2012) “Activator Induced Spread of Poly(ADP-Ribose) Polymerase Promotes Nucleosome Loss at Hsp70.” Mol Cell, 45:64-74. (PubMed)
- Ardehali BM, Yao J, Adelman K, Fuda N, Petesch S, Webb WW and Lis JT (2009) “Spt6 enhances the elongation rate of RNA polymerase II in vivo.” EMBO J 28(8):1067-77. Epub 2009 Mar 12. (PubMed)
- Petesch SJ and Lis JT (2008) “Rapid, Transcription-Independent Loss of Nucleosomes over a Large Chromatin Domain at Hsp70 Loci.” Cell 134: 74–84.
- Adelman K et al. (2006) “Drosophila Paf1 modulates chromatin structure at actively transcribed genes.” Mol Cell Biol. 26(1):250-60. (PubMed)
- Adelman K et al. (2005) “Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.” Mol Cell. 17(1):103-12. (PubMed)
Fast-acting conditional mutants
A rapidly-acting conditional mutant can be an invaluable tool in assessing the mechanistic role of a particular transcription factor in vivo. We are using a variety of fast-acting conditional mutants that encode Drosophila proteins that we hypothesize are critical for both establishing the potentiated promoter and for its activation. We are assessing the roles of these proteins in regulating chromatin architecture and function of the heat shock promoter in the seconds or minutes following the conditional disruption of a particular protein.
- Guzman E & Lis JT (1999) “Transcription factor TFIIH is required for promoter melting in vivo.” Mol Cell Biol. 19(8):5652-8. (PubMed)
Small molecule drugs
Drugs that rapidly inhibit particular active sites of proteins believed critical for transcription and its regulation are being used to evaluate the primary functions of these proteins in regulating chromatin architecture and function of the heat shock promoter.
- Ni Z et al. (2008) “P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo.” Mol Cell Biol. 28(3):1161-70. (PubMed)