A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development.

A Genetic Screen Identifies PRP18a, a Putative Second Step Splicing Factor Important for Alternative Splicing and a Normal Phenotype in Arabidopsis thaliana.

Splicing of pre-mRNA involves two consecutive trans-esterification steps that take place in the spliceosome, a large dynamic ribonucleoprotein complex situated in the nucleus. In addition to core spliceosomal proteins, each catalytic step requires step-specific factors. Although the Arabidopsis thaliana genome encodes around 430 predicted splicing factors, functional information about these proteins is limited. In a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana, we identified a mutant impaired in putative step II factor PRP18a, which has not yet been investigated for its role in pre-mRNA splicing in plants. Step II entails cleavage at the 3' splice site accompanied by ligation of the 5' and 3' exons and intron removal. In the prp18 mutant, splicing of a U2-type intron with non-canonical AT-AC splice sites in GFP pre-mRNA is reduced while splicing of a canonical GT-AG intron is enhanced, resulting in decreased levels of translatable GFP mRNA and GFP protein. These findings suggest that wild-type PRP18a may in some cases promote splicing at weak, non-canonical splice sites. Analysis of genome-wide changes in alternative splicing in the prp18a mutant identified numerous cases of intron retention and a preponderance of altered 3' splice sites, suggesting an influence of PRP18a on 3' splice site selection. The prp18a mutant featured short roots on synthetic medium and small siliques, illustrating that wild-type PRP18a function is needed for a normal phenotype. Our study expands knowledge of plant splicing factors and provides foundational information and resources for further functional studies of PRP18 proteins in plants.

A Genetic Screen for Pre-mRNA Splicing Mutants of Arabidopsis thaliana Identifies Putative U1 snRNP Components RBM25 and PRP39a.

In a genetic screen for mutants showing modified splicing of an alternatively spliced GFP reporter gene in Arabidopsis thaliana, we identified mutations in genes encoding the putative U1 small nuclear ribonucleoprotein (snRNP) factors RBM25 and PRP39a. The latter has not yet been studied for its role in pre-messenger RNA (pre-mRNA) splicing in plants. Both proteins contain predicted RNA-binding domains and have been implicated in 5' splice site selection in yeast and metazoan cells. In rbm25 mutants, splicing efficiency of GFP pre-mRNA was reduced and GFP protein levels lowered relative to wild-type plants. By contrast, prp39a mutants exhibited preferential splicing of a U2-type AT-AC intron in GFP pre-mRNA and elevated levels of GFP protein. These opposing findings indicate that impaired function of either RBM25 or PRP39a can differentially affect the same pre-mRNA substrate. Given a prior genome-wide analysis of alternative splicing in rbm25 mutants, we focused on examining the alternative splicing landscape in prp39a mutants. RNA-seq experiments performed using two independent prp39a alleles revealed hundreds of common genes undergoing changes in alternative splicing, including PRP39a itself, a second putative U1 snRNP component PRP40b, and genes encoding a number of general transcription-related proteins. The prp39a mutants displayed somewhat delayed flowering, shorter stature, and reduced seed set but no other obvious common defects under normal conditions. Mutations in PRP39b, the paralog of PRP39a, did not visibly alter GFP expression, indicating the paralogs are not functionally equivalent in this system. Our study provides new information on the contribution of PRP39a to alternative splicing and expands knowledge of plant splicing factors.

Chemical analysis of C-reactive protein synthesized by human aortic endothelial cells under oxidative stress.

C-reactive protein (CRP) is a clinical biomarker of inflammation, and high levels of CRP correlate with cardiovascular disease. The objectives of this study were to test our hypothesis that oxidized low-density lipoprotein (ox-LDL) induces the release of CRP from human aortic endothelial cells (HAECs) and to optimize several analytical methods to identify CRP released from cultured cells in a model of atherogenic stress. HAECs were incubated with copper-oxidized LDL, and the supernatant was subsequently purified by diethylaminoethyl chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified an optimal buffer for the elution of CRP, which contained 0.05 M sodium phosphate and 2.0 M NaCl (pH 4.5). Purified CRP was digested with trypsin and subjected to high-performance LC with an optimal mobile phase of acetonitrile-water containing 0.1% formic acid (50:50, v/v) and an optimal mobile phase flow rate of 0.2 mL/min. We identified optimal parameters for MS/MS analysis of CRP, including sheath gas pressure (80 psi), capillary temperature (275 °C), collision energy (25%), tube lens offset (-5 V), auxiliary gas pressure (0 psi), and isolation width of parent ion (m/z value = 3). Characterization of CRP was based on the extracted ion chromatograms and selected multiple-reaction monitoring spectra of three peptides (peptide-1, -2, and -3) derived from trypsin-digested intact CRP standard. CRP peptide-2 and peptide-3 were identified in the supernatant of ox-LDL-treated HAECs. Confirmation of CRP was based on LC-MS/MS and enzyme-linked immunosorbent assay analysis of CRP in purified HAEC supernatant, as well as real-time PCR analysis of CRP mRNA levels in HAECs.

Micellar electrokinetic chromatography profiles of human high-density lipoprotein phospholipids.

A simple and fast micellar electrokinetic chromatography (MEKC) method was developed to investigate phospholipids isolated from human high-density lipoproteins (HDL). To optimize the MEKC conditions, several factors including bile salt concentration and organic modifier concentration in the separation buffer as well as temperature have been examined. The optimal separation buffer chosen was a mixture of 50 mM bile salts, 30% v/v 1-propanol and 10 mM sodium phosphate (pH 8.5). The applied voltage and temperature selected were 25 kV and 40°C, respectively. Meanwhile, high-salt stacking has been performed for sample pre-concentration to enhance peak sensitivity. Several factors including organic modifier concentration and salt concentration in the sample matrix as well as sample injection time have been optimized. The optimal sample buffer selected was a mixture of 100 mM NaCl and 20% 1-propanol, and the optimal sample injection time selected was 32 s under a pressure of 0.5 psi. Several phospholipid standards including lysophosphatidyl choline, phosphatidyl choline (PC), sphingomyelin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and phosphatidic acid have been studied using the optimal MEKC method. The MEKC profile of the mixed phospholipid standards showed good separation and reproducibility. The linear ranges for PC and sphingomyelin were 0.025-1.2 and 0.025-2.0 mg/mL, respectively. The concentration limits of detection of PC and sphingomyelin were 0.0156 and 0.0199 mg/mL, respectively. Using phosphatidic acid as an internal standard, precision and accuracy have been measured for PC and sphingomyelin. The intraday and interday quantitative analysis showed good results. The new MEKC method has been used to characterize native, in vitro oxidized and glycated human HDL phospholipids within 16 min. At absorbance 200 nm, two similar peaks were observed for native and oxidized HDL phospholipids, but three peaks were observed for glycated HDL phospholipids. Interestingly, at absorbance 234 nm, distinctively different MEKC profiles were observed for the three HDL phospholipids.

Spontaneous regression of Kikuchi lymphadenopathy with oligoclonal T-cell populations favors a benign immune reaction over a T-cell lymphoma.

To aid in the initial diagnosis of Kikuchi lymphadenitis and to assess whether the composition of the T cells might shed light on the pathogenesis, we used nested polymerase chain reaction tests followed by high-resolution gel electrophoresis to determine the pattern of T-cell antigen receptor rearrangement in 56 consecutive cases. Except for 1 unusual case with recurrent lymphadenopathy, none had a monoclonal beta or gamma rearrangement. Eight cases had a polyclonal pattern at both beta and gamma loci, 20 cases had a mixed polyclonal beta and oligoclonal gamma pattern, and 27 cases had an oligoclonal pattern at both loci. The high frequency of oligoclonality did not indicate an early-stage T-cell lymphoma in evolution, as confirmed by spontaneous resolution of the lymphadenopathy in all cases within 6 months. Rather, it is consistent with reports of oligoclonal T cells in a variety of immune reactions. We conclude that, in the vast majority of cases, absence of a monoclonal T-cell receptor rearrangement excludes the possibility of T-cell lymphoma, and the presence of an oligoclonal pattern implies a benign immune reaction.