Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs

Abstract

Transcription start sites are critical switches for converting recognition of chromosomal DNA into active synthesis of RNA. Their functional importance suggests that they may be ideal targets for regulating gene expression. Here, we report potent inhibition of gene expression by antigene RNAs (agRNAs) complementary to transcription start sites within human chromosomal DNA. Silencing does not require methylation of DNA and differs from all known mechanisms for inhibiting transcription. agRNAs overlap DNA sequences within the open complex formed by RNA polymerase, and silencing is acutely sensitive to single base shifts. agRNAs effectively silence both TATA-less and TATA-box-containing promoters. Transcription start sites occur within every gene, providing predictable targets for agRNAs. Potent inhibition of multiple genes suggests that agRNAs may represent a natural mechanism for controlling transcription, may complement siRNAs and miRNAs that target mRNA, and will be valuable agents for silencing gene expression.

Evolution, biogenesis and function of promoter-associated RNAs

“…However, it is equally possible that tiRNAs and other PARs have evolved to serve an active role in the regulation of transcription, or in the communication of the event of transcription to regulate other processes associated with it. There is a growing body of work showing that exogenous small RNAs can activate or suppress transcription through epigenetic changes to promoter loci, [35][36][37][38][39][40][41][42][43] and a recent study has shown that synthetic PASRs sense or antisense to the c-MYC TSS reduced MYC mRNA levels. 10 Antisense non-coding RNAs overlapping promoters, perhaps produced by the bi-directional activity of RNAPII discussed above, may act as a constitutive "off switch" which tiRNAs counteract and silence, to "turn on" a gene.…”

RNA and transcriptional modulation of gene expression

“…However, recent studies have revealed that RNA mediated TGS is operative in human cells, and that the observed silencing is the result of RNA directed methylation of lysine residues 9 and 2 on histone 3 (H3K9 and H3K27, respectively), and DNA methylation at the targeted promoter. [40][41][42][43][44][45] However, DNA methylation may not be a conserved feature in all cases of small RNA mediated TGS in human cells, or may vary with the duration of exposure to the small RNA.…”

“…50 This model seems most plausible when small RNAs are targeted to RNAPII start sites, or TATAA, subsequently resulting in a small RNA/DNA interaction and robust transcriptional silencing. 42 However, silencing of regions upstream of the transcriptional start site or TATAA also result in gene silencing but perhaps this silencing is operative via a different mechanism involving an RNA-RNA mediated interaction (Fig. 1A).…”

Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs

“…6 In the case of mammalian cells, there is genetic evidence for the existence of DNA methylation guided by a siRNA-like subfamily of small RNAs called piRNAs, 7 and there are numerous reports on the existence of siRNA-guided chromatin remodeling. [8][9][10][11][12][13][14][15] Here, we gain insight into the mechanisms involved in siRNA-guided chromatin remodeling and use this knowledge to examine the existence of miRNA-guided chromatin remodeling.…”