Riboactivators: transcription activation by noncoding RNA

Abstract

The paradigm of gene regulation was forever changed by the discovery that short RNA duplexes could directly regulate gene expression. Most regulatory roles attributed to noncoding RNA were often repressive. Recent observations are beginning to reveal that duplex RNA molecules can stimulate gene transcription. These RNA activators employ a wide array of mechanisms to up-regulate transcription of target genes, including functioning as DNA-tethered activation domains, as coactivators and modulators of general transcriptional machinery, and as regulators of other noncoding transcripts. The discoveries over the past few years defy "Moore's law" in the breath-taking rapidity with which new roles for noncoding RNA in gene expression are being revealed. As gene regulatory networks are reconstructed to accommodate the influence of noncoding RNAs, their importance in maintenance of cellular health will become increasingly apparent. In fact, a new generation of therapeutic agents will focus on modulating the function of noncoding RNA.

Figure 1

Modular architecture of transcription activators. (A) Eukaryotic transcription factors typically contain a DNA binding domain (DBD) and an activation domain (AD). Separating the two domains eliminate transcriptional activation of target genes. However, tethering the AD to a DBD, by non-covalent protein interactions (X and Y) suffices to stimulate transcription. This principle is the basis of the 2-hybrid assay. In the 3-hybrid assay, a third bridging molecule tethers the AD to the DBD. In this case the bridging molecule is a bifunctional RNA molecule that bears and MS2-binding hairpin and a region that interacts with an RNA binding protein (RBP). (B) The DNA bound MS2 protein is used to tether RNA molecules bearing an MS2-binding hairpin and a putative RNA-Activation Domain (in red) upstream of a reporter gene. Three types of riboactivators are illustrated below.

Figure 2

Targets of protein and RNA activation domains. (A) Protein based transcription factors recruit several different protein complexes to gene promoters. These include several different chromatin modifying and remodeling complexes, co-activators, general transcription factors (GTFs), and the RNA polymerase II (Pol II). Nucleosomes are indicated as yellow spheres and the transcription start site denoted by a right-angled arrow. (B) An artificial riboactivator may interact with components of the transcriptional machinery, protein-based transcription factors and chromatin remodeling complexes. (C) The nascent transcript from the HIV-1 long terminal repeat (TAR) interacts with a viral protein transcription factor (TAT) and together they recruit pTEFb to a stalled polymerase. The pTEFb-associated kinase phosphorylates Pol II and promotes transcript elongation. (D) Developmental transcription factors (TF) like Dlx2 and NRSF/REST interact with their RNA partners to recruit the transcriptional machinery to their target genes.

Figure 3

RNA co-activators. (A) Upon binding their ligand, steroid receptors (SR) interact with several co-activators. A non-coding RNA (SRA) in complex with a protein (SRC-1) functions as a co-activator for steroid receptor class of transcription factors. Sub-elements of the RNA (in red) are important for SR mediated transcription activation. The co-activators interface with the transcriptional machinery and recruit it to the target gene promoter. (B) A general transcription factor complex (TFIIH) is often targeted by protein-based transcription factors, including steroid receptor class of TFs. TFIIH is found to be associated with the U1 snRNA. The RNA molecule is critical for the kinase activity and it strongly facilitates transcription reinitiation by Pol II.

Figure 4

RNA activation (RNAa) by natural or artificial RNA duplexes. (A) Naturally encoded RNA microRNAs (miRs) are processed into duplexes by Dicer and a single stranded ‘guide’ is loaded on to Ago2. The synthetic or artificial RNA duplexes, designed to target gene promoters also associate with Ago2. The RNA-Ago2 complex can stimulate (+) or repress (−) mRNA synthesis based on the precise sequence complementarity of its guide RNA. In addition to the bona fide mRNA (wavy black line) several non-coding sense and antisense transcripts are also generated at active promoters (wavy blue lines). (B) Three proposed models for the RNAa function. In the first, RNA hybridizes to the non-template strand leaving the template strand exposed for Pol II binding. The second model suggests that non-productive transcripts are eliminated by Ago-RNAa, thereby enhancing mRNA synthesis. The third model suggests that Ago-RNAa complex is transiently tethered to the PR gene promoter by hnRNP-K protein. This interaction facilitates the displacement of the repressive chromatin-binding protein HP-1γ from the promoter onto the antisense RNA. The promoter is then accessible to Pol II whereas the HP-1γ bearing RNA is likely released from the promoter.