Structural basis of a small molecule targeting RNA for a specific splicing correction.

Splicing modifiers promoting SMN2 exon 7 inclusion have the potential to treat spinal muscular atrophy, the leading genetic cause of infantile death. These small molecules are SMN2 exon 7 selective and act during the early stages of spliceosome assembly. Here, we show at atomic resolution how the drug selectively promotes the recognition of the weak 5' splice site of SMN2 exon 7 by U1 snRNP. The solution structure of the RNA duplex formed following 5' splice site recognition in the presence of the splicing modifier revealed that the drug specifically stabilizes a bulged adenine at this exon-intron junction and converts the weak 5' splice site of SMN2 exon 7 into a stronger one. The small molecule acts as a specific splicing enhancer cooperatively with the splicing regulatory network. Our investigations uncovered a novel concept for gene-specific alternative splicing correction that we coined 5' splice site bulge repair.

Effect of urea fertilization on growth of broad bean (Vicia faba L.) under various nickel (Ni) levels with or without acetic acid addition, using 15N-labeled fertilizer.

Although nickel (Ni) has direct relationship with nitrogen metabolism of plants, the high dose of Ni fertilizer in broad bean plants may affect the nitrogen use efficiency (NUE), impair plant development and even cause Ni pollution in soil. Thus, a pot experiment was set up to study the effect of urea fertilization on N-uptake, root and shoots' Ni content as well as growth of broad bean plants under different levels of Ni, using 15N tracer technique. 15N-labeled urea (5% 15N atom excess) was added at three doses (0, 30 and 60 mg N kg-1 soil). Nickel sulfate (NiSO4) was also applied at three levels (0, 50 and 100 mg Ni kg-1 soil). The experiment was laid out with or without acetic acid in randomized complete block design in three replicates. Treatment with the addition of 60 mg N + 50 mg Ni showed the highest values in dry weights of root and shoots, N-uptake by shoots, nitrogen derived from fertilizer (Ndff %) and NUE % by shoots in both with or without acetic acid solution. Higher rate of Ni addition can decrease shoot and root biomass by inhibiting the ability of the plant to uptake the nitrogen efficiently. However, addition of acetic acid solution induced the improvement of NUE % and Ndff % by shoot and root of broad bean plants. This study provides insight into how to improve plant yield without damaging the soil health and will be helpful to create a better world with sustainable agriculture.

Minimally Invasive Treatment of Ankle Fractures in Patients at High Risk of Soft Tissue Wound Healing Complications.

The complex nature of ankle fractures is magnified when seen in patients at high risk of soft tissue wound healing complications. The major categories include associated soft tissue injury, diabetes, tobacco use, peripheral vascular disease, malnutrition, alcoholism, and corticosteroid use. Because of the potential for wound dehiscence and infection with open reduction and internal fixation of ankle fractures in these patients, minimally invasive procedures have been described. The aims of the present study were to assess the possibility for, and evaluate the results and complications of, minimally invasive techniques for different types of malleolar fractures in high-risk patients. We report the clinical results of 47 high-risk patients who presented with malleolar fractures from January 2007 to December 2012 and underwent minimally invasive reduction and fixation. One patient (0.5%) developed a superficial infection; however, none of the patients displayed wound dehiscence or deep infection. Five patients (10.6%) required open reduction because of intraoperative failure to achieve anatomic reduction. Using the American Orthopaedic Foot and Ankle Society ankle-hindfoot scale, 15 of the patients (36%) treated with minimally invasive techniques experienced an excellent outcome. In contrast, 23 patients (55%) had a good, 3 (7%) a fair, and 1 (2.5%) a poor outcome. The results of our study have shown that minimally invasive fixation appears to be a satisfactory method for the management of malleolar fractures in high-risk patients and could be helpful in the avoidance of the complications associated with conventional open reduction and internal fixation.

Characterization of the RNA recognition mode of hnRNP G extends its role in SMN2 splicing regulation.

Regulation of SMN2 exon 7 splicing is crucial for the production of active SMN protein and the survival of Spinal Muscular Atrophy (SMA) patients. One of the most efficient activators of exon 7 inclusion is hnRNP G, which is recruited to the exon by Tra2-ß1. We report that in addition to the C-terminal region of hnRNP G, the RNA Recognition Motif (RRM) and the middle part of the protein containing the Arg-Gly-Gly (RGG) box are important for this function. To better understand the mode of action of hnRNP G in this context we determined the structure of its RRM bound to an SMN2 derived RNA. The RRM interacts with a 5'-AAN-3' motif and specifically recognizes the two consecutive adenines. By testing the effect of mutations in hnRNP G RRM and in its putative binding sites on the splicing of SMN2 exon 7, we show that it specifically binds to exon 7. This interaction is required for hnRNP G splicing activity and we propose its recruitment to a polyA tract located upstream of the Tra2-ß1 binding site. Finally, our data suggest that hnRNP G plays a major role in the recruitment of the Tra2-ß1/hnRNP G/SRSF9 trimeric complex to SMN2 exon 7.

Isolated pseudo-RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition.

Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the ß-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.

Rapid adsorption of benzotriazole onto oxidized carbon cloth as an easily separable adsorbent.

A commercial carbon cloth (CC) was oxidized by HNO3 acid and the features of the plain and oxidized CC were evaluated. The results of characterization illustrated that HNO3 oxidization duplicated the oxygen-containing functional groups and the surface area of the CC. The adsorption performance of the plain and oxidized CC (Oxi-CC) toward benzotriazole (BTR) was compared. The results disclosed that the uptake of BTR by oxidized CC was greater than the plain CC. Thence, the affinity of oxidized CC toward BTR was assessed at different conditions. It was found that the adsorption was quick, occurred at pH 9 and improved by adding NaCl or CaCl2 to the BTR solution. The kinetic and isotherm studies revealed that the surface of Oxi-CC is heterogeneous and the adsorption of BTR follows a physical process and forms multilayer over the Oxi-CC surface. The regenerability and reusability study illustrated that only deionized water can completely regenerate the Oxi-CC and that the Oxi-CC can be reused for five cycles without any loss of performance. The high maximum adsorption capacity of Dubinin-Radushkevich isotherm model (252 mg/g), ease of separation and regeneration, and maintaining the adsorption capacity for several cycles revealed the high efficiency and economical and environmental feasibility of Oxi-CC as an adsorbent for BTR.

RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing.

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

Structure of SRSF1 RRM1 bound to RNA reveals an unexpected bimodal mode of interaction and explains its involvement in SMN1 exon7 splicing.

The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.

Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7.

HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.

Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers.

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.

Preparation and characterization of humic acid-carbon hybrid materials as adsorbents for organic micro-pollutants.

The present work involves the preparation of novel adsorbent materials by the insolubilization and hybridization of humic acid (HA) with carbon. The prepared materials were characterized by N2 adsorption, elemental analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, solid-state (13)C cross polarization magic angle spinning nuclear magnetic resonance, and low-field nuclear magnetic resonance (NMR) relaxometry on wetted samples. The water solubility of these materials and the lack of effect of oxidants were also confirmed. With this background, the adsorption capacities toward phenol, 2,4,6-tricholrophenol, and atrazine were evaluated, using these as model compounds for organic micropollutants of concern in water. Experimental results show that the prepared materials are mesoporous and have a higher surface area than humic acid and even than the porous carbon in the case of carbon coating. They retain the basic features of the starting materials with lowered functional group content. Moreover, there are interesting new features. NMR relaxometry shows that equilibration of water uptake is very fast, making use in water simple. They have higher adsorption capacities than the pure materials, and they can be applied under a wide range of environmental conditions.