Puf proteins are developmental regulators that control mRNA stability and translation by binding sequences in the 3' untranslated regions of their target mRNAs. We have determined the structure of the RNA binding domain of the human Puf protein, Pumilio1, bound to a high-affinity RNA ligand. The RNA binds the concave surface of the molecule, where each of the protein's eight repeats makes contacts with a different RNA base via three amino acid side chains at conserved positions. We have mutated these three side chains in one repeat, thereby altering the sequence specificity of Pumilio1. Thus, the high affinity and specificity of the PUM-HD for RNA is achieved using multiple copies of a simple repeated motif.

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.

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.

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.protein design ͉ Puf proteins ͉ protein-RNA interaction ͉ adenosine-uracil-rich elements S pecific nucleotide sequences in mRNA molecules, often in noncoding sequences, regulate processes such as turnover, translation, localization, and splicing that are necessary for producing correct protein products in the right amounts at the right times and places. Proteins that bind to the specific RNA sequences often direct these posttranscriptional regulatory events, which are important during both embryonic development and cellular homeostasis. Two examples are the regulatory roles of sequences in the 3Ј UTRs of mRNAs and near alternative splice sites in pre-mRNAs. Expression of TNF␣ is regulated by an adenosine-uracil-rich element (ARE) in the 3Ј UTR of its mRNA, a common motif that confers instability on the message (1, 2). This sequence is recognized by the tristetraprolin (TTP) family of proteins, which initiates degradation of the AREcontaining mRNA (3). Overproduction of TNF␣, as in the absence of TTP, results in inflammatory disorders such as rheumatoid arthritis, cachexia, and autoimmunity (4). Alternative splicing events are directed by specific exon-intron junction motifs and positive and negative regulatory sequences within the exons or introns. Mutations in these sequences can result in splicing defects that lead to diseases such as cancer, spinal muscular atrophy, and cystic fibrosis (5).Because of the importance of RNA regulatory sequences in human health and disease, RNA-binding proteins with designed specificity could be important as tools for understanding processes directed by specific RNA sequences or perhaps as therapeutic agents. For example, an RNA-binding protein with specificity for a splicing regulatory sequence could induce a preferred alternatively spliced product by blocking an alternative splicing event. Or an RNA-binding protein with specificity for a particular RNA could be tracked by fusing the RNA-binding domain with a green fluorescent protein partner. Other work on designing RNA-binding proteins has focused on folded RNA targets such as the HIV-1 Rev response element RNA by using zinc fingers or arginine-rich motif peptides and relied generally on screening of proteins with selected randomized positions or building a chimeric molecule (6-12). Our previous studies of the Puf family protein, human Pumilio1, a sequence-specific RNAbinding protein, suggested that it could be used ...

Zinc finger proteins are generally thought of as DNA-binding transcription factors; however, certain classes of zinc finger proteins, including the common C(2)H(2) zinc fingers, function as RNA-binding proteins. Recent structural studies of the C(2)H(2) zinc fingers of transcription factor IIIA (TFIIIA) and the CCCH zinc fingers of Tis11d in complex with their RNA targets have revealed new modes of zinc finger interaction with nucleic acid. The three C(2)H(2) zinc fingers of TFIIIA use two modes of RNA recognition that differ from the classical mode of DNA recognition, whereas the CCCH zinc fingers of Tis11d recognize specific AU-rich sequences through backbone atom interaction with the Watson-Crick edges of the adenine and uracil bases.

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have ‘high-energy’ phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature’s most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF3− transition-state mimic. We describe the enzyme’s conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

SUMMARY Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from pre-microRNA. 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.

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.