Joel M. Gottesfeld and Carlos F. Barbas III
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 USA

Correspondence: Joel M. Gottesfeld; and Carlos F. Barbas III
joelg@scripps.edu

Two recent reports demonstrate that in vivo selection can isolate novel RNAs that activate transcription when
tethered to a gene promoter. This highlights the structural plasticity that allows RNA to fulfill many functions
normally carried out by proteins.

The chemical biology community has long sought to devise methods to control gene expression by targeting
specific DNA sequences with novel peptides, oligonucleotides, or small molecules [3–5]. Natural or synthetic
DBDs have been tethered to peptides derived from potent viral activators [6–9], and fully synthetic peptides
have been fused to natural DBDs for gene activation [10, 11]. In another approach, peptide libraries have been
screened by phage display methods for sequences that will bind to particular coactivators. An excellent example of this is reported by Frangioni et al., who screened phage libraries for peptides that bound CBP/p300 [12]. The selected peptides then can be fused to DBDs to affect gene activation. Perhaps the most surprising finding of these studies is the diversity of workable solutions to the problem of gene activation, particularly in the role played by the AD.

Two pioneering groups have taken a novel approach toward generating synthetic activators where the activation domain is composed entirely of RNA [13, 14]. In the June issue of Chemistry & Biology, David Liu and colleagues present the results of a functional screen for RNA activators in yeast. Three separate components are required for this method (see Figure 1 in Buskirk et al. [14]). First, selection depends upon a reporter plasmid harboring the consensus binding site for the LexA DBD (the LexA operator), a minimal promoter, and a selectable marker such as the HIS3 gene (or ?-galactosidase for quantitation). Second, an expression plasmid encoding a fusion of the LexA DBD to the coat protein of the MS2 RNA bacteriophage is introduced into the same cells, and third, a plasmid encoding a random sequence of 40 or 80 nucleotides (N40 or N80) fused to additional RNA-coding sequences and two copies of the MS2 RNA hairpin-coding sequence (this hairpin RNA binds the MS2 coat protein). This entire expression system is driven by a yeast RNA polymerase III promoter. These plasmids are introduced into yeast cells, which are then selected for growth on media lacking histidine. To stabilize the random RNA region from intracellular degradation, this region is embedded within a larger RNA sequence that is known to have a stable secondary structure. In order to activate transcription, the LexA-MS2 fusion protein must first bind to the LexA operator and then recruit MS2 RNA linked to an activating RNA sequence. Successful RNAs will then recruit the transcription apparatus, resulting in strong transcription of the reporter gene. Expression of the HIS3 gene product allows survival of yeast cells on selection media lacking histidine and recovery of RNA sequences that activated transcription for further study. Potent activating RNAs were indeed isolated, some of which activated transcription at levels comparable to natural ADs, such as yeast Gal4 and herpes virus VP16.

In a first round of screening, about 0.2% of the clones from the N40 library passed initial selection and rescreening (compared to only 0.01% of the N80 library), and, remarkably, one clone actually activated transcription more than 10-fold relative to a Gal4-positive control. Control experiments indicated that tethering of the activating RNA to the reporter template via the MS2 protein-RNA interaction is essential for gene activation. When the sequences of the selected RNAs were determined, a variety of structural motifs could be predicted based on computer modeling. This suggests that either various RNA structures can serve as activators by targeting common components of the transcription apparatus or that multiple protein targets can be bound by various RNA structures.

Random mutations (at a level of 20% per base) were introduced into one of the most potent activator RNA
sequences, and this RNA was subjected to an additional round of selection under more stringent conditions,
yielding 32 unique sequences. One of these activated transcription 53-fold stronger than the Gal4 control AD and only 2-fold weaker than the highly potent VP16 AD. Thus, evolution generated an RNA activator comparable to one of the most efficient known natural protein ADs. In another recent report by Ptashne and colleagues [13], a random sequence loop of only 10 nucleotides attached to an RNA stem was used for selection, and far lower levels of gene activation were found compared to the larger 40 nucleotide sequence used in the present study [14]. Additional experiments by Liu et al. [14] compared the sequences of the evolved RNAs, showing that several structural elements in the selected RNA could play key roles in transcriptional activation. Lastly, mutagenesis of one of the most potent RNA activators revealed the important structural components of this RNA required for activation, most notably the importance of base-paired regions of the RNA.

While the molecular identity of the targets of the selected activating RNAs has yet to be determined, it is
reasonable to speculate that these RNAs bind their targets (coactivators or general transcription factors) with
comparable affinities to natural transcriptional ADs. Once further investigation has identified these target
molecules, it will be of interest to compare the mechanisms by which the natural protein activators and the RNAs bind the same or similar targets. Successful identification of RNA transcriptional activators, along with aptamers and ribozymes, illustrates the vast structural and functional diversity available to RNA, perhaps even rivaling the diversity found in protein structures.

References:

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10. Gerber, H.P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S., and Schaffner, W. (1994). Science 263, 808-811.

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12. Frangioni, J.V., LaRiccia, L.M., Cantley, L.C., and Montminy, M.R. (2000). Nat. Biotechnol. 18, 1080-1085.

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14. Buskirk, A.R., Kehayova, P.D., Landrigan, A., and Liu, D.R. (2003). Chem. Biol. 10, 533-540. 

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