Peter A. Beal

Small molecule inhibitors of the RNA-dependent protein kinase

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the alpha subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

Chemical modification of siRNA bases to probe and enhance RNA interference

Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely, those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.

Novel Modifications in RNA

The past several years have seen numerous reports of new chemical modifications for use in RNA. In addition, in that time period, we have seen the discovery of several previously unknown naturally occurring modifications that impart novel properties on the parent RNAs. In this review, we describe recent discoveries in these areas with a focus on RNA modifications that introduce spectroscopic tags, reactive handles, or new recognition properties.

Controlling protein activity with ligand-regulated RNA aptamers.

Controlling the activity of a protein is necessary for defining its function in vivo. RNA aptamers are capable of inhibiting proteins with high affinity and specificity, but this effect is not readily reversible. We describe a general method for discovering aptamers that bind and inhibit their target protein, but addition of a specific small molecule disrupts the protein-RNA complex. A SELEX protocol was used to raise RNA aptamers to the DNA repair enzyme, formamidopyrimidine glycosylase (Fpg), and neomycin was employed in each round to dissociate Fpg-bound RNAs. We identified an RNA molecule able to completely inhibit Fpg at 100 nM concentration. Importantly, Fpg activity is recovered by the addition of neomycin. We envision these ligand-regulated aptamers (LIRAs) as valuable tools in the study of biological phenomena in which the timing of molecular events is critical.

Solid-phase synthesis of acridine-based threading intercalator peptides

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding.

Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domains dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

ADAR Proteins: Structure and Catalytic Mechanism

Since the discovery of the adenosine deaminase (ADA) acting on RNA (ADAR) family of proteins in 1988 (Bass and Weintraub, Cell 55:1089-1098, 1988) (Wagner et al. Proc Natl Acad Sci U S A 86:2647-2651, 1989), we have learned much about their structure and catalytic mechanism. However, much about these enzymes is still unknown, particularly regarding the selective recognition and processing of specific adenosines within substrate RNAs. While a crystal structure of the catalytic domain of human ADAR2 has been solved, we still lack structural data for an ADAR catalytic domain bound to RNA, and we lack any structural data for other ADARs. However, by analyzing the structural data that is available along with similarities to other deaminases, mutagenesis and other biochemical experiments, we have been able to advance the understanding of how these fascinating enzymes function.

N2-Modified 2-aminopurine ribonucleosides as minor-groove-modulating adenosine replacements in duplex RNA

Nucleoside analogs that project substituents into the minor groove when incorporated into duplex RNA perturb the binding of proteins and can affect base pairing specificity. The synthesis of 2-aminopurine ribonucleoside analogs and their phosphoramidites, their incorporation into duplex RNA, their postsynthetic modification via Cu-catalyzed azide-alkyne cycloaddition (CuAAC), and their effect on duplex stability and base pairing specificity are described.

The binding site of the RNA-dependent protein kinase (PKR) on EBER1 RNA from Epstein–Barr virus

The RNA‐dependent protein kinase (PKR) is an interferon‐induced, RNA‐activated enzyme that phosphorylates the eukaryotic initiation factor 2α, rendering the translation machinery inactive. Viruses have developed strategies for preventing the action of PKR, one of which is the production of small RNAs that inhibit the enzyme. Epstein–Barr virus (EBV) encodes EBER1, a 167 nucleotide non‐coding RNA that is constitutively expressed by the EBV‐infected cells. EBER1 binds PKR in vitro and has been shown to prevent inhibition of translation by PKR in vitro. We used affinity cleavage by the EDTA·Fe‐modified double‐stranded RNA‐binding domain (dsRBD) of PKR to show that stem–loop IV (nucleotides 87–123) of EBER1 makes specific contacts with the dsRBD. To further demonstrate the specificity of this interaction, we generated a deletion mutant of EBER1, comprising only stem–loop IV (mEBER1). Cleavage patterns produced on mEBER1 by the bound dsRBD were remarkably similar to those found on full‐length EBER1. Using cleavage data from two different dsRBD mutants, we present a model of the interaction of PKR dsRBD and mEBER1.