Traci M. Tanaka Hall

Size Selective Recognition of siRNA by an RNA Silencing Suppressor

RNA silencing in plants likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene silencing. Tombusviruses express a 19 kDa protein (p19), which has been shown to suppress RNA silencing in vivo and bind silencing-generated and synthetic small interfering RNAs (siRNAs) in vitro. Here we report the 2.5 A crystal structure of p19 from the Carnation Italian ringspot virus (CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays that CIRV p19 protein acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.

Crystal Structure of a Hedgehog Autoprocessing Domain: Homology between Hedgehog and Self-Splicing Proteins

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.

Structural basis for recognition of AU-rich element RNA by the HuD protein

Hu proteins bind to adenosine-uridine (AU)-rich elements (AREs) in the 3′ untranslated regions of many short-lived mRNAs, thereby stabilizing them. Here we report the crystal structures of the first two RNA recognition motif (RRM) domains of the HuD protein in complex with an 11-nucleotide fragment of a class I ARE (the c-fos ARE; to 1.8 Å), and with an 11-nucleotide fragment of a class II ARE (the tumor necrosis factor α ARE; to 2.3 Å). These structures reveal a consensus RNA recognition sequence that suggests a preference for pyrimidine-rich sequences and a requirement for a central uracil residue in the clustered AUUUA repeats found in class II AREs. Comparison to structures of other RRM domain–nucleic acid complexes reveals two base recognition pockets in all the structures that interact with bases using residues in conserved ribonucleoprotein motifs and at the C-terminal ends of RRM domains. Different conformations of nucleic acid can be bound by RRM domains by using different combinations of base recognition pockets and multiple RRM domains.

Crystal Structure of a Pumilio Homology Domain

Puf proteins regulate translation and mRNA stability by binding sequences in their target RNAs through the Pumilio homology domain (PUM-HD), which is characterized by eight tandem copies of a 36 amino acid motif, the PUM repeat. We have solved the structure of the PUM-HD from human Pumilio1 at 1.9 A resolution. The structure reveals that the eight PUM repeats correspond to eight copies of a single, repeated structural motif. The PUM repeats pack together to form a right-handed superhelix that approximates a half doughnut. The distribution of side chains on the inner and outer faces of this half doughnut suggests that the inner face of the PUM-HD binds RNA while the outer face interacts with proteins such as Nanos, Brain Tumor, and cytoplasmic polyadenylation element binding protein.

Engineering RNA sequence specificity of Pumilio repeats

Puf proteins bind RNA sequence specifically and regulate translation and stability of target mRNAs. A “code” for RNA recognition has been deduced from crystal structures of the Puf protein, human Pumilio1, where each of eight repeats binds an RNA base via a combination of three side chains at conserved positions. Here, we report the creation of seven soluble mutant proteins with predictably altered sequence specificity, including one that binds tightly to adenosine-uracil-rich element RNA. These data show that Pumilio1 can be used as a scaffold to engineer RNA-binding proteins with designed sequence specificity.

Engineering splicing factors with designed specificities

Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.

Phosphate and R2D2 Restrict the Substrate Specificity of Dicer-2, an ATP-Driven Ribonuclease

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs (miRNAs) from pre-miRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage and an N-terminal helicase motif, whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs.

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the “core” PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a −2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.