Abstract IA11: Control of gene expression in the nucleus by small RNAs

Double-stranded RNAs are widely used to control gene expression and are a promising class of therapeutic agent. Most studies have focused on recognition of mRNA in the cytoplasm and inhibition of translation. My presentation focuses on the action of small RNAs in the nucleus to modulate transcription or redirect alternative splicing. In 2005, my laboratory observed that synthetic RNAs complementary to the promoter region at the transcriptional start site of human progesterone receptor (PR) could block PR expression [[1][1]]. Inhibition was potent and robust, consistent with the suggestion that the synthetic RNAs were exploiting an endogenous mechanism for controlling gene expression. Promoter-targeted synthetic miRNAs were also capable of robust gene silencing [[2][2]], further supporting the existence of endogenous regulatory pathways. Modulation required complete seed sequence complementarity to the PR promoter, consistent with a mechanism that involves RNA-RNA recognition through Watson-Crick base-pairing. In 2007, we observed that small RNAs could also target the PR gene promoter and activate PR expression [[3][3]]. Activation was observed in cells that had a low, but detectable, level of PR expression, while gene silencing had been observed in cells with a high level of PR expression. Inactive and active RNAs compete for recognition, demonstrating that small changes in target site location affect activity. These results are reminiscent of protein transcription factors, which bind to specific sequences, are sensitive to target site location or small conformation changes, and can activate gene expression in some contexts but not others. Our promoter-targeted RNAs bind to long noncoding RNA transcripts at the PR promoter [[4][4]]. Use of antisense oligonucleotides to reduce transcript levels also reduces RNA-mediated modulation of gene expression. The small RNAs recruit AGO2 to the noncoding transcript, and inhibition of AGO2 expression reverses gene silencing or activation [[5][5]]. While AGO2 is normally expected to cleave fully complementary targets in the cytoplasm, our data suggests that AGO2 does not cleave transcripts associated with gene promoters in the nucleus. A noncoding RNA transcript also overlaps the 3′ terminus of the PR gene [[6][6]]. Small RNAs complementary to this noncoding transcript modulate PR expression, depending on the cell line used. The PR gene loops, juxtaposing its 3′ and 5′ termini and providing a direct path for a signal transmitting a signal from the 3′ terminus of the gene to the promoter. We have investigated whether RNAs can modulate other nuclear events. We designed small RNAs to be complementary to sequence within exons or introns near splice sites. These RNAs redirect alternative splicing and AGO2 was recruited to pre-mRNA [[7][7]]. These data provide more support for the action of small RNA in cell nuclei and for the conclusion that AGO2 may not always cause RNA cleavage when it encounters complementary targets in the nucleus. Our data suggest that small RNAs can bind noncoding transcripts and modulate gene expression. Binding occurs in proximity to the gene promoter, in cis relative to the target gene. The RNA-AGO complex acts like a transcription factor to affect the transcription machinery and tip the balance of inducible genes like PR towards repression or activation. Citation Format: David Corey. Control of gene expression in the nucleus by small RNAs [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA11. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7