RNA interference (RNAi) involves silencing gene expression through recognition of mRNA by small duplex RNAs (1
). Some recent reports have suggested that RNAs complementary to gene promoters can inhibit (2–9
) or activate (10–14
) gene expression in mammalian cells. In contrast to duplex RNAs that recognize mRNA and act post-transcriptionally, RNAs that target gene promoters modulate gene transcription. We describe RNAs that target gene promoters as antigene RNAs (agRNAs) to distinguish them from traditional siRNAs that target and cleave mRNA.
There is no evidence that promoter-targeted RNAs directly interact with chromosomal DNA. Instead, they have been reported to bind to non-coding RNA transcripts that overlap gene promoters (8
). Three studies have proposed that small duplex RNAs associate with non-coding RNAs that are transcribed in the sense orientation (i.e. the same direction as mRNA) (8
). Our laboratory identified an antisense transcript as the molecular target for agRNAs that modulate expression of the PR gene (16
). This PR antisense transcript initiates within the coding region of the gene and spans ~70
000 bases upstream from the transcription start site.
Our approach for further understanding how agRNAs bind to non-coding transcripts and alter transcription from gene promoters involves examining the potential role of RNA-binding proteins that facilitate RNA/RNA interactions. We reasoned that studying the function of the argonaute (AGO) family of proteins provided a logical starting point since members of this family are critical components in the RNAi pathway.
There are four AGO proteins (AGO1–4) in humans. AGO2 is the ‘catalytic engine’ of RNAi, responsible for recognition of mRNA and subsequent cleavage of the transcript (18–21
). AGO2 has also been suggested to be involved in miRNA biogenesis (22
). Using a minimal in vitro
system AGO1 and AGO2 have been shown to possess the ability to dissociate miRNA duplexes, while AGO3 and AGO4 do not (23
). In another report, reintroduction of any AGO variant into embryonic stem (ES) cells deficient for expression of all four AGO variants rescues miRNA silencing defects and reduces apoptosis, suggesting that AGO3 and AGO4 can assist RNAi (24
). Functional redundancy of AGO has also been inferred from mRNA or miRNA pull-down experiments showing detection of similar bound transcripts regardless of which AGO variant is being isolated (20
). Finally, all four human AGO proteins exhibit similar preferences for binding to duplex RNA with mismatches at different positions, although only AGO2 efficiently unwound fully complementary duplexes (26
). Taken together, these data demonstrate a role for AGO2 in these RNA-mediated processes, but also suggest that AGO1, AGO3 and AGO4 proteins may be involved in these mechanisms.
For AGO proteins to alter promoter activity, they must be located within the cell nucleus. Although AGO proteins primarily reside in the cytoplasm, studies have indicated that they are also found in the nucleus (27–31
). In Caenorhabditis elegans
an AGO protein NRDE-3 was found to be required for nuclear siRNA import (27
). In mammalian cells, nuclear activity of AGO was first inferred from the observation of potent gene silencing of small nuclear RNA 7SK (28
). A highly specific anti-AGO2 antibody was subsequently used to identify AGO2 in nuclear lysate (29
) and fluorescence correlation and cross-correlation spectroscopy also revealed nuclear AGO2 (30
). Most recently, importin-8 has been reported to be involved in the translocation of AGO2 from cytoplasm to nucleus (31
There have been multiple reports on the role of AGO proteins in the mechanism of promoter-targeted RNAs. One laboratory has implicated AGO2 in RNA-mediated gene activation (10
). Our laboratory reported that either AGO1 or AGO2 might be necessary for gene silencing (32
), while other reports determined AGO1, along with other non-AGO proteins, as critical using multiple experimental approaches (9
). However, few reports investigated a role for AGO3 or AGO4 in either gene silencing or activation (10
). Here we investigate involvement of human AGO1–4 in agRNA-mediated gene silencing or activation of PR expression. Using multiple experimental strategies we find that AGO2 protein is the best candidate for mediating both gene silencing and activation ().
Summary of data on participation of AGO variants in modulation of PR gene expression by agRNAs