Small RNAs Regulate Transcription by Interacting with Noncoding RNA Transcripts
Schwartz, Jacob C.
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General methods for controlling gene expression have long been appreciated as an attractive target in drug design. Recently, the Corey lab has demonstrated that short RNA duplexes designed to target the promoter region for human genes can inhibit or activate gene expression in a sequence dependent manner. The mechanism by which RNAs achieve promoter recognition has remained unclear. Sequence specific recognition could be achieved by (1) RNA hybridization to genomic DNA, or (2) RNA recognition of some uncharacterized RNA species. Promoter targeted duplex RNA has been shown to recruit argonaute proteins to the promoter DNA and these proteins are necessary for duplex RNAs to regulate transcription. Argonaute proteins are known to recognize RNA:RNA interactions. However, genes targeted with duplex RNAs have no characterized transcripts in their promoters. I tested the hypothesis that promoter RNA transcripts exist and serve as a substrate for short duplex RNAs to hybridize to and regulate gene expression of adjacent genes. I found previously undiscovered RNA transcripts expressed from the promoter of progesterone receptor (PR) using RT-PCR. Quantitative RT-PCR of the promoter RNA of PR reveals expression levels between 10 and 1000 fold lower than PR in T47D and MCF7 breast cancer cells. I have cloned three transcripts overlapping the promoter of PR from two cell lines – T47D and MCF7, each with unique splicing and transcription start sites. All of these transcripts initiate within the protein coding region of PR and run antisense to the gene PR. I have been able to show that the promoter transcripts can be immunoprecipitated with antibodies against the argonaute proteins in cells transfected with duplex RNAs targeting the promoter of PR but not in cells transfected with mismatched duplex RNAs. Also, biotinylated RNAs bind to and pull down these noncoding RNAs. Finally, knockdown of the antisense transcript with an antisense oligonucleotide prevent gene activation by duplex RNAs. Following this study, our lab uncovered that duplex RNAs can target beyond the 3' terminus of genes and silence or activate transcription. I further showed that this transcription regulation is mediated by argonaute binding to noncoding RNAs overlapping the 3' terminus of the genes, PR and BRCA1. The signal is transmitted from the 3' terminus to the gene promoter because the 5' and 3' ends of these genes are held in a chromatin loop, which I validated using a chromatin conformation capture assay. This brings the ends of the gene in close proximity to each other. Due to this interaction, short RNAs that bind a noncoding RNA at the 3' end of the gene also physically interacts with noncoding RNAs that associate with the gene promoter. This is confirmed by RNA immunoprecipitation of both transcripts with duplex RNAs targeting either the 5' or 3' ends of the gene. More than 20 years ago, it was found that proteins recognizing DNA at the 5’ end of genes could regulate transcription. This study presents a paradigm shift implicating noncoding RNAs at the 5' and 3' ends of genes can be recognized by proteins which activate or inhibit transcription of adjacent protein coding genes. Recent studies demonstrate an abundance of RNAs transcribed in human cells that do not code for protein. My results suggest a new model for duplex RNA recognition of gene promoters. Argonaute proteins loaded with one strand of the RNA duplex recognizes, through Watson-Crick base pairing, a noncoding transcript that is associated with chromatin at the promoter of the targeted gene. This RNA:RNA interaction in close proximity to the promoter mediates protein-protein interactions between argonaute and other factors on the promoter to turn off or on gene expression.