Haihong Wu

A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III

RNases III are a family of double‐stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN tetraloops, AGAA and AGUU, using NMR spectroscopy. Both tetraloops have the same overall structure, in which the backbone turn occurs on the 3′ side of the syn G residue in the loop, with the first A and G in a 5′ stack and the last two residues in a 3′ stack. A non‐bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various tetraloop sequences defines a novel family of RNA tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.

Identification and Solution Structure of a Highly Conserved C-terminal Domain within ORF1p Required for Retrotransposition of Long Interspersed Nuclear Element-1

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded β sheet that is packed against three α-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.

H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification

During the biogenesis of eukaryotic ribosomal RNA (rRNA) and spliceosomal small nuclear RNA (snRNA), uridines at specific sites are converted to pseudouridines by H/ACA ribonucleoprotein particles (RNPs). Each H/ACA RNP contains a substrate-specific H/ACA RNA and four common proteins, the pseudouridine synthase Cbf5, Nop10, Gar1, and Nhp2. The H/ACA RNA contains at least one pseudouridylation (ψ) pocket, which is complementary to the sequences flanking the target uridine. In this article, we show structural evidence that the ψ pocket can form the predicted base pairs with substrate RNA in the absence of protein components. We report the solution structure of the complex between an RNA hairpin derived from the 3′ ψ pocket of human U65 H/ACA small nucleolar RNA (snoRNA) and the substrate rRNA. The snoRNA–rRNA substrate complex has a unique structure with two offset parallel pairs of stacked helices and two unusual intermolecular three-way junctions, which together organize the substrate for docking into the active site of Cbf5. The substrate RNA interacts on one face of the snoRNA in the complex, forming a structure that easily could be accommodated in the H/ACA RNP, and explains how successive substrate RNAs could be loaded onto and unloaded from the H/ACA RNA in the RNP.

Structure Determination of Protein⧸RNA Complexes by NMR

Structure determination of protein?RNA complexes in solution provides unique insights into factors that are involved in protein/RNA recognition. Here, we review the methodology used in our laboratory to overcome the challenges of protein?RNA structure determination by nuclear magnetic resonance (NMR). We use as two examples complexes recently solved in our laboratory, the nucleolin RBD12/b2NRE and Rnt1p dsRBD/snR47h complexes. Topics covered are protein and RNA preparation, complex formation, identification of the protein/RNA interface, protein and RNA resonance assignment, intermolecular NOE assignment, and structure calculation and analysis.

Measurement of small scalar and dipolar couplings in purine and pyrimidine bases

A suite of spin-state-selective excitation (S3E) NMR experiments for the measurements of small one-bond (13C-13C, 15N-13C) and two-bond (1H-13C, 1H-15N) coupling constants in 13C,15N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between α and β sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S3E rather than α/β-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides.