64To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward 65 genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This 66 effort generated a collection of sixteen mutants impaired in various splicing-related proteins, 67 many of which had not been recovered in any prior genetic screen or implicated in splicing in 68 plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP 69 biogenesis pathway, transcription, and mRNA transport. We have described eleven of the 70 mutants in recent publications. Here we present the final five mutants, which are defective, 71 respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME 72WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing 74 data and analyses of differential gene expression and alternative splicing patterns for the cbp80 75 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, 76 for which such information was not yet available. Sequencing of small RNAs from the cbp80 77 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) 78 precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing 79 factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss 80 the cumulative findings and their implications for the regulation of pre-mRNA splicing 81 efficiency and alternative splicing in plants. The mutant collection provides a unique resource for 82 further studies on a coherent set of splicing factors and their roles in gene expression, alternative 83 splicing and plant development. 84 85 86 87 88 89 90 91 92 93 94 95Splicing of pre-mRNAs by the excision of introns and ligation of flanking exons is a 96 prerequisite for the expression of most eukaryotic genes. Splicing entails two transesterification 97 reactions carried out by the spliceosome, a large and dynamic ribonucleoprotein (RNP) machine 98 located in the nucleus. At least six structurally and functionally distinct spliceosomal complexes 99 containing core spliceosomal proteins, transiently-associated factors and different combinations 100 of five different small nuclear (sn) RNAs -U1, U2, U4, U5 and U6 -act sequentially to execute 101 the two catalytic steps of the splicing process (Matera and Wang, 2014;Yan et al., 2017). The 102 spliceosome is able to carry out constitutive splicing, in which the same splice sites are always 103 used for a given intron, and alternative splicing, in which splice site usage for a given intron is 104 variable. Alternative splicing increases transcriptome and proteome diversity (Nilsen and 105 Graveley, 2010;Syed et al., 2012;Reddy et al., 2013) and is important for development and 106 stress adaptation in plants (Staiger and Brown, 2013; Filichkin et al., 2015; Szakonyi and Duque, 107 2018). 108Most information on spliceosome composition and the splicing mechanism has been derived 109 from genetic, bioc...

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.

The cellular lineage of sinonasal T/NK (natural killer) cell lymphoma remains controversial. Lineage assignment is difficult because T cells and NK cells have a similar morphology and surface markers. Consequently, the assignment must depend heavily on the status of T-cell receptor (TCR) rearrangement. A monoclonal TCR rearrangement supports a T lineage; however, a corresponding monoclonality test for NK cells has not yet been established. Each NK cell bears a distinct set of killer cell immunoglobulin (Ig)-like receptors (KIRs) that are randomly distributed over three groups. In principle, restriction of the KIR repertoire signifies a monoclonal or possibly oligoclonal NK-cell proliferation, just as Ig light-chain restriction usually indicates a monoclonal B-cell neoplasm. Using a novel group-specific reverse transcriptase-polymerase chain reaction, we found a restricted KIR repertoire in most sinonasal lymphomas (9 of 10), but only rarely in T-cell lymphomas (2 of 10) or reactive conditions involving T/NK cells (1 of 10). KIR+ sinonasal lymphomas usually lacked a monoclonal TCR-gamma rearrangement pattern, expressed another NK cell receptor, NKG2a, and were usually CD56-positve, cyclin-dependent kinase-6 (CDK6)-positive, CD44-negative, a phenotype already reported to indicate a true NK cell lineage. We conclude that, although sinonasal lymphomas have heterogeneous genotypes and phenotypes, a restricted KIR repertoire without TCR-gamma rearrangement provides preliminary support for the monoclonality hypothesis and can be used for defining a true NK-cell lineage in a subset of sinonasal lymphomas.

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.

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.