Plant pre-mRNA splicing in fission yeast, Schizosaccharomyces pombe

Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the splicing machinery. We have analyzed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency of a plant gene AmA1 in Schizosaccharomyces pombe. Earlier, we reported the cloning of AmA1, a seed albumin gene from Amaranthus hypochondriacus [A. Raina, A. Datta, Proc. Natl. Acad. Sci. USA 89 (1992) 11774]. In the absence of an in vitro splicing system for plants, the expression of AmA1 genomic clone in S. pombe has been used to analyze splicing of intron constructs. We aim to focus on S. pombe as a possible alternative and examined its effectiveness as a host for plant gene splicing. The results show here that pre-mRNA transcripts of AmA1 gene underwent splicing in S. pombe

Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations

B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population

Inositol Polyphosphates Regulate Zebrafish Left-Right Asymmetry

SummaryVertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer’s vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca2+ flux during LR specification

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth▿ †

The inositol 1,3,4,5,6-pentakisphosphate (IP5) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP6) in eukaryotic cells. Previous studies have shown that IP6 is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1+ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1+ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP6 production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1+ function, encoding an IP6 kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1+ upstream of ipk1+. We propose that IP6 production is a key signaling linchpin for regulating multiple essential cellular processes

Premature termination of RNA polymerase II mediated transcription of a seed protein gene in Schizosaccharomyces pombe

The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3′-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT–PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3′-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3′-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611–949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene

MACS-enriched B cells were loaded with Indo-1-AM (5 μg/ml), and labeled with AA4

1, anti-CD23, and anti-HSA for B cell subset identification. Cells were applied to a cytometer, basal Ca levels were monitored for 90 s, then cells were stimulated with 10 μg/ml anti-IgM (indicated by the arrow) and monitored for 4-6 min; T1 (dotted black line), T2 (black line), mature FoB (gray line), MZ (dotted gray line). Data is a representative of 4-6 independent experiments. <p><b>Copyright information:</b></p><p>Taken from "Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations"</p><p></p><p>Biological Procedures Online 2007;9():73-83.</p><p>Published online Jan 2007</p><p>PMCID:PMC2275047.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2007 Biological Procedures Online.</p

Freshly isolated MACS-enriched wt B cells were fixed with 2% paraformaldehyde, permeabilized and labeled with antibodies directed against CD19, CD21, and HSA for B cell subset identification

B cells from (A) were examined for basal PIP levels utilizing an anti-PI(4,5)P antibody. Data is an example of multiple experiments. B cells were stimulated for the indicated times with 20 μg/ml anti-IgM, immediately fixed and permeabilized, then labeled and examined for PIP levels as in (B); T1 (filled diamonds), T2 (filled squares), mature FoB (open circles), preMZ (filled triangles), mature MZB (open triangles). Data is displayed as the average MFI of duplicate samples, and is a representative of at least 4 experiments. The specificity of the anti-PIP antibody was tested by pre-incubation of the antibody with PIP liposomes for 15 min prior to intracellular labeling of MACS-enriched total B cells as in (B). <p><b>Copyright information:</b></p><p>Taken from "Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations"</p><p></p><p>Biological Procedures Online 2007;9():73-83.</p><p>Published online Jan 2007</p><p>PMCID:PMC2275047.</p><p>Article © by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s). Copying, printing, redistribution and storage permitted. Journal © 1997-2007 Biological Procedures Online.</p

Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton's tyrosine kinase and phospholipase C-gamma2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes