5 and gene expression was controlled by the CMV promoter, the down-regulation of genes was not caused by promoter competition for erythroid-specific and ubiquitous transcription factors that are essential for globin gene expression. To further confirm the role of the intronic triplex-forming sequence in suppressing gene expression, we repeated these experiments using the same gene-expression vector, but with only the region coding for the 15-nt triplex-forming sequence deleted (pA/X-triplex-CMV6-XL5) (Figs. gene expression Abstract We have identified regulatory mechanisms in which an RNA transcript forms a DNA Rabbit Polyclonal to PKR duplex?RNA triple helix with a gene or one of its regulatory elements, suggesting potential auto-regulatory mechanisms in vivo. We describe an conversation at the human PP121 locus, in which an RNA segment embedded in the second intron of the gene forms a DNA?RNA triplex with the HS2 sequence within the locus control region, a major regulator of expression. We show in human K562 cells that this triplex is usually stable in vivo. Its formation causes displacement from HS2 of major transcription factors and RNA Polymerase II, and consequently in loss of factors and polymerase that bind to the human and promoters, which are activated by HS2 in K562 cells. This results in reduced expression of these genes. These effects are observed when a PP121 small length of triplex-forming RNA is usually introduced into cells, or when a full-length intron-containing human transcript is usually expressed. Related results are obtained in human umbilical cord blood-derived erythroid progenitor-2 cells, in which expression is usually similarly affected by triplex formation. These results suggest a model in which RNAs conforming to the strict sequence rules for DNA? RNA triplex formation may participate in feedback regulation of genes gene in human erythroid K562 cells. encodes a protein that is a fusion made up of fubi, a ubiquitin-like protein, and ribosomal protein S30. Although fubi function is usually unknown, posttranslational processing produces S30, a component of the 40S ribosome. We used this system to refine methods necessary to detect triplex formation and to distinguish it from R-loop formation, a potential source of confusion. We then applied these methods to search for other examples of DNARNA triplexes and identified an conversation between an RNA sequence present within an intron of the human adult PP121 gene and an upstream regulatory element within hypersensitive site 2 (HS2) of the locus control region (LCR). The effect of this interaction is usually to displace transcription factors from the regulatory site and affect expression of members of the family. This system represents a feedback mechanism in which a transcript could affect its own expression by forming a triple-strand structure at a nearby regulatory element. Results In Vivo Triplex-Forming RNA in K562 Cells: The Gene as a Source and Target. The methods we employed for detecting DNARNA triple-stranded structure formation in vitro are shown in (FAU ubiquitin-like and ribosomal PP121 protein S30 fusion), a proapoptotic regulatory gene that is expressed in K562 cells and down-regulated in human breast, prostate, and ovarian cancers (18C21). Our search showed that one of the more abundant RNAs satisfying the criteria for triplex formation corresponded in sequence to antisense transcript (Fig. 1gene as part of a canonical triplex. However, because it happens to be a palindromic sequence it could also form an R-loop in which the RNA partially displaces one of the DNA strands, and forms a heteroduplex with the other, while still maintaining a complex with three strands. We chose this gene as a way to develop methods for demonstrating triplex formation and eliminating heteroduplex formation as an explanation for our results. Open in a separate window Fig. 1. FAU-tfRNA forms triplex with gene dsDNA in vitro. (locus. Triplex-forming region is located at exon 5 (red bar) and palindromic antisense triplex-forming sequence (FAU-tfRNA) is usually underlined (red). (gene triplex region in vitro, and such a triplex PP121 is usually resistant to RNase H but subject to RNase A digestion. Cy3-labeled FAU dsDNA (green) and Cy5-labled FAU-tfRNA (red) were incubated as described (and (gene. FAU-tfRNA was transfected into K562 cells and its effect on gene expression was examined. Cy5-labeled.