Systematic screens of proteins binding to synthetic microRNA precursors.

We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag-specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre-miRNAs with phylogenetically conserved loop regions and 25 precursors of other well-characterized miRNAs for chemical synthesis in 3'-biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre-miRNAs, we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let-7 family, and that KSRP binds strongly to pre-miR-1-2.

Probing mechanism and transition state of RNA refolding.

Kinetics and the atomic detail of RNA refolding are only poorly understood. It has been proposed that conformations with transient base pairing interaction are populated during RNA refolding, but a detailed description of those states is lacking. By NMR and CD spectroscopy, we examined the refolding of a bistable RNA and the influence of urea, Mg(2+), and spermidine on its refolding kinetics. The bistable RNA serves as a model system and exhibits two almost equally stable ground-state conformations. We designed a photolabile caged RNA to selectively stabilize one of the two ground-state conformations and trigger RNA refolding by in situ light irradiation in the NMR spectrometer. We can show that the refolding kinetics of the bistable RNA is modulated by urea, Mg(2+), and spermidine by different mechanisms. From a statistical analysis based on elementary rate constants, we deduce the required number of base pairs that need to be destabilized during the refolding transition and propose a model for the transition state of the folding reaction.

NMR-spectroscopic characterization of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme.

In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.

Conformational dynamics of bistable RNAs studied by time-resolved NMR spectroscopy.

The structural transition between two alternate conformations of bistable RNAs has been characterized by time-resolved NMR spectroscopy. The mechanism, kinetics, and thermodynamics underlying the global structural transition of bistable RNAs were delineated. Both bistable RNA conformations and a partial unstructured RNA of identical sequence could be trapped using photolabile protecting groups. This trapping allowed for an investigation of the initial folding from an unfolded RNA to one of the preferred conformations of the bistable RNA and of the structural transitions involved. Folding of the secondary structure elements occurs rapidly, while the global structural transition of the bistable RNA occurs on a time scale of minutes and shows marked temperature dependence. Comparison of these results with bistable systems previously investigated leads to the prediction of activation enthalpies (DeltaH++) associated with global structural transitions in RNA.

Time-resolved NMR studies of RNA folding.

The application of real-time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second to minute regime. In addition, the initial investigations suggest that different folding rates are observed for one structural transition may be due to the hierarchical folding units of RNA. Many of the experiments developed in the field of NMR of protein folding cannot directly be transferred to RNA: hydrogen exchange experiments outside the spectrometer cannot be applied since the intrinsic exchange rates are too fast in RNA, relaxation dispersion experiments on the other require faster structural transitions than those observed in RNA. On the other hand, information derived from time-resolved NMR experiments, namely the acquisition of native chemical shifts, can be readily interpreted in light of formation of a single long-range hydrogen bonding interaction. Together with mutational data that can readily be obtained for RNA and new ligation technologies that enhance site resolution even further, time-resolved NMR may become a powerful tool to decipher RNA folding. Such understanding will be of importance to understand the functions of coding and non-coding RNAs in cells.

The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction.

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.

Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes.

We report an optimized synthesis of all canonical 2'-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology.

Kinetics of RNA refolding in dynamic equilibrium by 1H-detected 15N exchange NMR spectroscopy.

By implementing new NMR methods that were designed to map very slow exchange processes we have investigated and characterized the refolding kinetics of a thermodynamically stable 34mer RNA sequence in dynamic equilibrium. The RNA sequence was designed to undergo a topologically favored conformational exchange between different hairpin folds, serving as a model to estimate the minimal time required for more complex RNA folding processes. Chemically prepared RNA sequences with sequence-selective (15)N labels provided the required signal separation and allowed a straightforward signal assignment of the imino protons by HNN correlation experiments. The 2D version of the new (1)H-detected (15)N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of (15)N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau(obs) approximately T(1){(15)Nu} < 5 s) and a third fold which is static on this time scale. A 1D version of the (15)N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies E(a) and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies are comparable to values obtained for the slow refolding of RNA sequences of similar thermodynamic stability but less favorable topology.

Structure of PTB bound to RNA: specific binding and implications for splicing regulation.

The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two separated pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor.