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 B$^{act}$ spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation

Structural and Functional Investigation of Two Splicing Factors: Human hnRNP G and yeast Npl3

The genetic information stored in the DNA is transferred by a first step of transcription to RNA. The transcribed pre-messenger RNA (pre-mRNA) is subjected to a series of maturation steps before it is translated into functional protein. One of the major maturation steps is constitutive splicing, which consists in the removal of non-coding sequences (introns) within the RNA and the ligation of coding sequences (exons). Interestingly, the pre-mRNA can be spliced differently by the inclusion or exclusion of specific exonic or intronic elements resulting in different mRNAs from a single gene. This process known as alternative splicing allows the modulation of the message conveyed by each gene. The resulting diversity of messages provides eukaryotic cells some flexibility to react to variations in intra and extracellular conditions. Splicing is subjected to very tight regulation steps to ensure that the correct pieces of spliced RNA are ligated together and give a meaningful mRNA. This control is ensured by two major classes of proteins, namely serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). The splicing outcome will depend on their competition to bind the pre-mRNA at a precise position that will allow them to either stimulate or inhibit the splicing reaction. This action is achieved by influencing the recruitment of the nuclear machinery responsible for the splicing catalysis, the spliceosome. In the Spinal Muscular Atrophy (SMA) disease, patients lack a functional copy of Survival of Motor Neurons 1 (SMN1) gene and retain only a closely related gene SMN2 that does not allow the splicing of exon 7 leading to the production of truncated, and non-functional SMN proteins. One of the main activators of SMN2 exon 7 splicing is hnRNP G. We solved the NMR structure of the RNA Recognition Motif (RRM) of this splicing factor bound to an SMN2 derived RNA and showed, in combination with in vivo splicing assays, that contrary to what was previously proposed, this protein binds SMN2 at a specific site in exon 7. This discovery was important because it allowed us to propose a model of how hnRNP G and its interacting partner Tra2-b1 are co-recruited on SMN2 exon 7. Moreover, it allows the investigation of novel structure-based therapeutic strategies targeting those two strong splicing activators of SMN2 exon 7. In contrast to humans, only ~4% of Saccharomyces cerevisiae genes contain introns, and very few among them undergo alternative splicing. S. cerevisiae lacks classical SR proteins but contains 3 SR-like proteins. Npl3 is the only SR-like protein in budding yeast that was reported to influence splicing. We identified the minimal RNA motifs recognized by the 2 RRMs of Npl3 and determined the structure of their individual complexes using NMR. The structures explained the specific interaction of Npl3 with RNA and allowed us to design point mutations affecting the binding of either RRM1 or RRM2 to RNA. Using those Npl3 variants, we showed that the interaction of both domains with RNA is required for the splicing function of Npl3 in yeast. However, the binding of RRM1 but not RRM2 to RNA is required for the interaction with chromatin remodeling complexes. Finally, we used a combined CRAC and iCLIP approach to show that the motifs bound by each RRM in vitro were enriched in the Npl3 binding sites in vivo. Based on all those results, we proposed a model explaining the binding of both RRMs to the natural RNA targets of Npl3. In summary, we investigated both structurally and functionally how two important splicing factors belonging to the two main families of splicing regulators bind specifically their RNA target sequences and how this binding contributes to their functions in vivo

Effected Irradiated Sewage Sludge and Compost on Jatropha Yield Production and Using 15Nitrogen

Studies on irradiation of sewage sludge indicated its feasibility both economical and hygienically but work in this point is limited. Also the effect of non-irradiated and irradiated sewage sludge as a source of N on yield production and improvements fertility of sandy soil. In this research using the organic manure, the effects were increased soil fertility and crop yield. Aseeds yield production by Jatropha curcas L., these treatments can be arranged in the following descending order: T5> T6> T7> T4> T2> T3> T1. The best value of Ndff% recorded with rate 50% gamma irradiated sewage sludge + 50% ammonium sulphate fertilizer. Values of Ndfo % for 75.2%, 74.8% and 73.3% for (50% non-irradiated sewage sludge + 50% ammonium sulphate), (50% compost + 50% ammonium sulphate) and irradiated sewage sludge + 50% ammonium sulphate respectively. In general, the FUE% with 100% ammonium sulphate was ammonium sulphate alone lower than those recorded with ammonium sulphate plus none or irradiated sewage sludge and compost when T5, T7, T6 and T1 treatments were considered

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-β1. 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-β1 binding site. Finally, our data suggest that hnRNP G plays a major role in the recruitment of the Tra2-β1/hnRNP G/SRSF9 trimeric complex to SMN2 exon 7.ISSN:1362-4962ISSN:0301-561

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

Abstract 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

Effects of fertigation components on emitter clogging and soil infiltration rate

A field experiment was conducted to study the effect of fertigation components on emission uniformity, emitter clogging and soil infiltration rate. The effects of two types of emitters with two discharges and six fertilization treatments were undertaken. For all fertilization treatments, the emission uniformity significantly decreased while emitters clogging ratio increased with increasing number of fertigations which were periodically applied throughout the 180-day season. The 4 Lh-1 discharge emitters resulted in less clogging than the 2 Lh-1 emitters. Clogging problems obtained with the pressure compensating (PC) emitters was less than those of normal emitters. The maximum clogging ratios 29.42% and 28.72% at the end of 180days season were recorded for the plots received ammonium sulfate and humic acid+ ammonium sulfate fertigation respectively, on using normal type emitters of 2 Lh-1 discharge.Finally, Humic acid treatment showed a clear reduction in infiltration rate from 14.60 mm min-1 to 6.80 mm min-1

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.ISSN:2050-084