Having it both ways: transcription factors that bind DNA and RNA

120

172

Retinoic acid (RA) plays important roles in development by modulating gene transcription through nuclear receptor activation. Increasing evidence supports a role for RA and RA receptors (RARs) in synaptic plasticity in the brain. We have recently reported that RA mediates a type of homeostatic synaptic plasticity through activation of dendritic protein synthesis, a process that requires dendritically localized RARα and is independent of transcriptional regulation. The molecular basis of this translational regulation by RA/RARα signaling, however, is unknown. Here we show that RARα is actively exported from the nucleus. Cytoplasmic RARα acts as an RNA-binding protein that associates with a subset of mRNAs, including dendritically localized glutamate receptor 1 (GluR1) mRNA. This binding is mediated by the RARα carboxyl terminal F-domain and specific sequence motifs in the 5′UTR of the GluR1 mRNA. Moreover, RARα association with the GluR1 mRNA directly underlies the translational control of GluR1 by RA: RARα represses GluR1 translation, while RA binding to RARα reduces its association with the GluR1 mRNA and relieves translational repression. Taken together, our results demonstrate a ligand-gated translational regulation mechanism mediated by a non-genomic function of RA/RARα signaling.

Section: Rar␣ Regulates the Translation Of A Reporter Construct Contamentioning

confidence: 99%

Retinoic acid-gated sequence-specific translational control by RARα

Poon¹,

Chen

2008

120

172

“…To accommodate the entire protein-binding surface, the major groove of the RNA helix extends into the duplex formed by the internal loop residues. The cross-strand stacked guanines G 8 , G 22 , and G 23 form the expanded major groove. The bases at the major groove facing the stack along one side are G 18 , G 19 , U 20 , U 21 , G 22 , G 23 , and G 8 .…”

Section: Tetraloops (15 16) a 16 Is Flipped Out Of The Loop And Is mentioning

confidence: 99%

“…5a). K241, the only residue contributed by the domain linker segment, directly contacts the carbonyl oxygen of G 22 . A 11 (in a pair with U 21 ) donates a hydrogen bond to the carboxylate oxygen of E60.…”

Section: Tetraloops (15 16) a 16 Is Flipped Out Of The Loop And Is mentioning

confidence: 99%

Crystal structure of NF-κB (p50) ₂ complexed to a high-affinity RNA aptamer

Huang

Cassiday

et al. 2003

158

157

We have recently identified an RNA aptamer for the transcription factor NF-B p50 homodimer [(p50) 2 ], which exhibits little sequence resemblance to the consensus DNA target for (p50) 2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR͞RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem-loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3 helix, together with the major groove of the irregular 3 helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the B DNA͞p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50) 2.

“…The occurrence of apparent natural RNA decoys (23) suggests the possibility of expressing therapeutic decoy RNAs in cells to modulate gene expression (24). Such RNA decoys might offer certain advantages over RNA-based knockdown strategies that require many cellular components for RNA processing and activity [i.e., RNA interference technologies (25)].…”

Section: Discussionmentioning

confidence: 99%

Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

Cassiday

Maher

2003

In vitro-selected RNA aptamers are potential inhibitors of diseaserelated proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-B. This RNA aptamer competitively inhibits DNA binding by NF-B in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-B in the eukaryotic nucleus. Selection for improved binding to NF-B from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific ''decoy'' activity, inhibiting transcriptional activation by its NF-B target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.T he artificial modulation of gene expression is a major goal of modern disease therapeutics. Recently, RNA aptamers have been investigated as inhibitors of disease-related proteins (reviewed in ref. 1). In vitro selections using large combinatorial RNA libraries have made trivial the identification of RNA sequences that bind with high affinities to target macromolecules. However, it is not intuitive that an RNA molecule selected for binding in vitro likewise will bind and inhibit the target protein within the complex environment of a living eukaryotic cell. Despite this caveat, some RNA aptamers identified through in vitro selection have been shown to perform the desired inhibitory function when expressed in cells. For example, Famulok and colleagues (2) used in vitro selection to identify an RNA aptamer that inhibits the intracellular domain of the ␤2 integrin LFA-1. A second example of an in vitro-selected RNA aptamer that subsequently was shown to function in vivo is an inhibitory aptamer identified by Lis and colleagues (3) against the Drosophila B52 splicing protein.We have been intrigued by the possibility of identifying an RNA ''decoy'' for a model transcription factor, human NF-B. Such a decoy molecule might fold so as to bind at or near the DNA-binding site of the transcription factor, sequestering NF-B and reducing its ability to activate target genes. A member of the rel homology protooncogene family of proteins, NF-B exists in cells primarily as a homodimer composed of two p50 subunits [(p50) 2 ] or a heterodimer composed of one p50 and one p65 subunit (p50͞p65). Both forms of NF-B bind similar sites in duplex DNA [e.g., 5Ј-GGGACTTTCC (4)] but appear to activate transcription through distinct mechanisms because, unlike p65, p50 lacks a potent carboxyl-terminal transcription activation domain (5). Inhibition of NF-B activity by the controlled expression of an RNA decoy potentially could have important thera...