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 alternativesplicing 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 a-helix and does not involve the β-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 a-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

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

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

The hnRNP family: insights into their role in health and disease

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies

Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1due to predominant skipping of exon 7. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes

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

Magnetohydrodynamic boundary layer flow past a stretching plate and heat transfer

The present work is concerned with unsteady two-dimensional laminar flow of an incompressible, viscous, perfectly electrically conducting fluid past a nonisothermal stretching sheet in the presence of a transverse magnetic field acting perpendicularly to the direction of fluid. By means of the successive approximation method, the governing equations for momentum and energy have been solved. The effects of surface mass transfer fω, Alfven velocity α, Prandtl number P, and relaxation time parameter τ0 on the velocity and temperature have been discussed. Numerical results are given and illustrated graphically for the problem considered

Long-Term Outcome of Orthotopic Neobladder Reconstruction after Radical Cystectomy – Sohag Experience

Objective: To assess the long-term outcome of neobladder reconstruction after radical cystectomy. Patients and Methods: In this retrospective study we evaluated the records of 90 patients (70 males and 20 females) subjected to radical cystectomy and orthotopic neobladder reconstruction at Sohag University Hospital, Sohag, Egypt, between January 1999 and January 2006. The age of the patients ranged from 35 to 70 years with a median age of 42 years. All patients had invasive bladder carcinoma: squamous cell carcinoma in 55, transitional cell carcinoma in 33 and adenocarcinoma in 2 patients. Thirty-five patients had a W-neobladder with serous-lined extramural ureteral reimplantation, 35 patients had Studer pouch and 20 patients had colonic (sigmoid) neobladder reconstruction. After surgery all patients were followed up for a period of 6 to 84 months (median 45 months) in order to evaluate the functional and oncological outcome. Results: Thirty (33.3%) patients developed early complications (defined as within 30 days from surgery). Chest infection and wound infection occurred in 3 (3.3%) and 5 (5.6%) patients, respectively, and was treated with antibiotics. Eight patients developed paralytic ileus which was managed with naso-gastric tube drainage. Re-operation was required in 8 patients: 3 required re-suturing of the abdominal incision, bleeding occurred in 2, while 2 developed intestinal obstruction and one ureterointestinal leakage. Pouch leakage was observed in 6 patients. Late complications occurred in 18 (20%) patients. Re-operation was necessary in 15 cases: 4 with stone formation in the neobladder, 3 with incisional hernia, 5 with a stricture at the ureterovesical junction, one with vesicourethral stricture and 2 with vesicovaginal fistula. Complete continence during day and night was achieved in 63 patients (70%). The daytime continence rate was 86.6% (87 patients), while 24 patients (26.7%) had nocturnal incontinence and 3 patients (3.3%) were fully incontinent. Four patients (4.4%) used clean intermittent self-catheterization. Twenty-seven (30%) patients developed recurrence of cancer within 4 to 30 months from surgery, among them 3 with urethral recurrence, 20 with local pelvic recurrence and 4 with distant metastases. All 27 patients with tumor recurrence died from disease progression during the follow-up period; 4 patients died from causes not related to the operation. Conclusion: Orthotopic neobladder reconstruction provides acceptable continence rates and has an acceptable early and late complication rate. We therefore recommend that all suitable patients undergoing cystectomy should have an option of orthotopic neobladder reconstruction. Keywords: orthotopic diversion, neobladder, cystectomyAfrican Journal of Urology Vol. 13 (3) 2007: pp. 198-20