Molecular recognition of single-stranded RNA: Neomycin binding to poly(A)

Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A₃₀) with a K_d in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47 °C. The poly(A) duplex, which melts at 44 °C (pH 5.5), was observed to melt at 61 °C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.     

Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.     

A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.     

Molecular recognition of nucleic acids: coralyne binds strongly to poly(A)

The small molecule coralyne was found to bind preferentially and strongly to single-stranded poly(A) with an apparent association constant (Ka) of (1.8+/-0.3) x 10(6)M(-1). Binding of coralyne to poly(A) is predominantly enthalpically driven with a stoichiometry of one coralyne per four adenine bases. Poly(A) forms a coralyne dependent secondary structure with a melting temperature of 60 degrees C, for the conditions of our study.     

Molecular recognition by Kluyveromyces of amphotericin B-loaded, galactose-tagged, poly (lactic acid) microspheres

In an effort to develop a new way of drug delivery, especially for polyenic antifungal molecules, we have incorporated amphotericin B (AmB) into biodegradable galactosylated poly (L-lactic acid) (L-PLA) and poly (L-lactic-co-glycolic acid) (PLGA) microspheres. These drug carriers were prepared by solvent evaporation using an oil/water (o/w) emulsion. The ratio of galactosyl spacers with different chain lengths was 1.74-2.78%. The maximal quantity of AmB encapsulated reported to 100 mg of the galactosylated microspheres was 7.14 mg for L-PLA (encapsulation rate 45% of mole) and 6.42 mg for PLGA derivatives (encapsulation rate 81% of mole). In our yeast model, drug release depended on three factors: (i) presence of galactosylated antennae, (ii) length of galactosyl antenna and (iii) nature of the polymer. More of the AmB trapped in PLGA microspheres was released than from PLA microspheres. These novel functionalised microspheres could be required for the delivering of therapeutic agents according to their recognition to specific cells.     

Pattern recognition by TREM-2: binding of anionic ligands

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.     

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.     

Kinetic study of cooperative binding reaction of toluidine blue to poly (alpha,L-glutamic acid) by means of the electric-field pulse method.

Interaction of toluidine blue with helical and randomly coiled poly(α,L -glutamic acid) was studied with absorption spectra, titration, and electric-field pulse measurements. The obtained values of various parameters for the helical form of poly(α,L -glutamic acid)differed from those for its coiled form. The difference of parameters in these two forms of poly(α,L -glutamic acid) was attributed to differences of the binding mechanism in both forms. Furthermore, the binding of toluidine blue to poly(α,L -glutamic acid) was considered to be due to hydrogen binding in the helical conformation and ionic interaction in the coiled conformation of the polymer.     

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.     

Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules

Self-structure induction in single stranded poly(A) has been one typical example of the various ways that could be used to modulate nucleic acid structural aspects through binding of small molecules. For the first time, the interaction between a series of small molecules and poly(A) has been investigated to understand the nature of the structural features in DNA binding small molecules that could be responsible for the formation of self-structure in single stranded poly(A) molecules. Classical intercalators like ethidium, coralyne, quinacrine and proflavine, partial intercalators like berberine and palmatine and classical minor groove binders like hoechst 33258 and DAPI have been chosen for this study. The binding of each of these molecules to poly(A) has been characterized by absorption spectral titration, job plot and isothermal titration calorimetry. Self-structure formation was monitored from circular dichroic melting, optical melting and differential scanning calorimetry. The results revealed that while all the intercalators studied induced self-structure formation, partial intercalators did not induce the same in poly(A). Of the two classical DNA minor groove binding molecules investigated, hoechst was effective in inducing self-structure while DAPI was ineffective. Self-structure induction in poly(A) was observed to be directly linked to the cooperative binding of the molecules to poly(A) in that all the molecules that bound cooperatively induced self-structure in poly(A). Structural and thermodynamic aspects of the interaction leading to self-structure formation are described.     

Inhibition of tristetraprolin deadenylation by poly(A) binding protein

Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.     

Stereospecific analysis of triglycerides: an alternative method

A new method for the stereospecific analysis of triglycerides is demonstrated on a corn oil which had been analyzed by an earlier method. The triglyceride (1,2,3-triacyl L-glycerol) was partially degraded by means of methyl magnesium bromide. The 1,3-diglyceride was then isolated, and converted into l-2-phosphatidyl phenol, from which phospholipase A cleaved the fatty acids from position 1. The remaining lysophosphatide was analyzed to yield the fatty acids at position 3 of the original triglyceride. The fatty acid composition of position 2 was determined after hydrolysis of the corn oil with pancreatic lipase. This method has the advantage over the one reported previously of yielding direct analyses of the fatty acids in each of the three positions.     

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).     

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.     

A kinetics and modeling study of RANTES(9-68) binding to heparin reveals a mechanism of cooperative oligomerization

Heparan sulfate (HS) and heparin bind to virtually all chemokines and have been shown to play critical roles in the regulation of their activities. However, both binding mechanisms and structural features involved in chemokine-HS interactions remain poorly defined. In the study presented here, we analyzed the binding of heparin to RANTES(9-68), a N-terminally truncated form of the CC-chemokine RANTES. Using biochemical and surface plasmon resonance (BIAcore system) approaches, we showed that the RANTES(9-68)-heparin interaction was characterized by a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity. Since RANTES(9-68) remains monomeric in solution, we concluded that heparin induced chemokine dimerization. The structure of a complex involving a RANTES dimer and a heparin heptadecasaccharide was proposed by molecular modeling. This model was used to design a dimer of "head to head" coupled octasaccharides that would fit the internal symmetry of the chemokine dimer. This engineered oligosaccharide bound RANTES(9-68) much better than a natural heparin fragment of the same length, further supporting the interaction process and the proposed structural model. Altogether, the data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions.     

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.