Exon-Skipping Responsiveness of Fourteen Endogenous Exons in Cortical Cultures During Depolarization Treatment

<p>Gel panels represent RT-PCR analysis of endogenous exons with gene and exon numbers as indicated above each panel. Each panel represents 24-h treatment of cortical cultures with 0, 25, and 50 mM KCl (lanes, left to right). One PCR primer in each sample was 5′ end labeled with ³²P. Raw exon inclusion values (EI) and change in exon inclusion values (ΔEI; boldface) are given below each panel. E8 of MEN1 and E2 of actin are constitutive exons; all other exons are alternative cassette exons. A single asterisk (*) indicates 6-h KCl treatment.</p

Test of Inhibitors of NMDA Receptors and Signaling Pathways on the CI Cassette Exon-Skipping Response in Excited Neurons

<div><p>(A) Schematic summarizes the experimental procedure: cortical cultures were transfected with CaMKII_22 CI wt0, and pre-incubated with inhibitor at final concentrations as indicated just above gel panels. With the inhibitor remaining in the culture medium, KCl was then added (25 or 50 mM) to induce depolarization. Gel panels and graph in (A) show effects of NMDA receptor antagonists MK801 and AP5. Pre-inc, pre-incubated.</p> <p>(B) Gel panels and graph shows the effects of cell-permeable inhibitors, KT5720, H89, and KN93. Experiments were performed as described for (A). The exon-included (<<) and -skipped (<) mRNAs are indicated.</p></div

Nucleotide Sequence Requirements for KCl-Induced Depolarization Effects on Splicing in Primary Neurons

<div><p>(A) Splicing silencer requirement for the induced response. Splicing reporters based on CI wt0 were expressed in primary neurons via the CaMKII_22 promoter, and depolarization was induced by addition of KCl as in the experiment of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-g003" target="_blank">Figure 3</a>. Structures of splicing reporters are identical except for point mutations in silencing motifs as shown in the schematic. UAGG silencing motifs at positions 51 and 93 of the CI cassette exon and a GGGG motif in the downstream intron are shown for the wild-type substrate, CI wt0. Mutations in splicing reporters, E8, E9, E17, D0, and T8, are underscored. All splicing reporters [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-b025" target="_blank">25</a>] were subcloned and expressed from the CaMKII_22 promoter. Graph displays effects of depolarization on the splicing of the wild-type and mutant substrates. RT-PCR analysis was performed with forward primer (1) and the common reverse primer as for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-g003" target="_blank">Figure 3</a>. A representative gel panel is shown. Exon-included (>>) and exon-skipped (>) mRNAs are indicated. ΔEI values are shown.</p> <p>(B) Effects of exonic splicing enhancers. Point mutations in six exonic enhancers of the CI cassette exon were tested as described in (A). Sequence and position of SC35, ASF/SF2, and SRp40 motifs are shown at left; mutations within each motif are underscored. Effects of mutations as a function of depolarization in neurons and ΔEI values are shown.</p> <p>SD, standard deviation.</p></div

Cell-Specific Splicing Reporter Assay for Depolarization Induced Alternative Splicing and Effects on Cortical Neuron Viability

<div><p>(A) Changes in C1 cassette exon inclusion in response to depolarization were measured by transfecting CaMKII_22 and Gfa2 C1 wt0 splicing reporters into day 5 (DIV) cortical cultures, followed by addition of KCl (25 mM or 50 mM) into the culture medium for 6 or 24 h to induce depolarization. Inset summarizes order of addition. Control cultures were mock treated in parallel (0 mM KCl). Splicing patterns were determined from RNA harvested from the samples by RT-PCR using forward primers (rightward arrows) specific for transcripts expressed from CaMKII_22 (1) and Gfa2 (2), together with a common reverse primer specific for the C2 exon (leftward arrow). Endogenous transcripts were amplified similarly with a forward primer (E) together with the C2-specific primer. A minimum number of three measurements were used for each mean and standard deviation shown. Structures of the splicing reporters are shown; exon numbers are based on the human GRIN1–006 transcript (schematic, top). Bar graphs represent percent C1 exon inclusion at two time points (6 and 24 h) after addition of KCl. Representative gels are shown (bottom). Effects on splicing reporters, (1) CaMKII_22 (lanes 7–12) and (2) Gfa2 (lanes 13–18), are shown with those of the corresponding endogenous (E) <i>GRIN1</i> mRNA (lanes 1–6). Induced changes in splicing (ΔEI, %) were calculated as described above (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-g001" target="_blank">Figure 1</a>). The exon-included (<<) and -skipped (<) mRNAs are indicated. SD, standard deviation.</p> <p>(B) Viability of CaMK-expressing cortical neurons following KCl-mediated depolarization. Cortical neurons were transfected with CaMKII_22 EYFP plasmid, and treated under mock or depolarization conditions (0 and 50 mM KCl, respectively) as described for (A). Cultures were stained with trypan blue and more than 200 EYFP-expressing neurons were scored for trypan blue staining. The percentage of viable cells for each condition is shown; <i>n</i> = total cells scored. Representative neurons from these cultures are shown. Left panels of each condition show CaMKII-EYFP fluorescence. Right panels show bright field (trypan blue) and fluorescence together.</p></div

Reversibility and Effects of Neuronal Differentiation in the Response of the CI Cassette Exon to KCl-Induced Depolarization in Rat Cortical Cultures

<p>Alternative splicing of the endogenous CI cassette exon (NMDA R1 transcript) was monitored in rat cortical cultures in response to KCl treatment beginning at day 6 (lanes 1–3), 8 (lanes 4–6), 12 (lanes 7–9), and 14 (lanes 10–12) after plating. Cell cultures were treated with 0, 25, or 50 mM KCl in the culture medium for 24 h. Reversibility of induced effects was monitored in day 12 cultures, which were treated with 0, 25, and 50 mM KCl for 24 h followed by washout into fresh medium without KCl for an incubation time of 0 (lanes 13–15), 6 (lanes 16–18), 12 (lanes 19–21), and 24 (lanes 22–24) h after washout. Relative quantities of exon-included (<<) and -skipped (<) mRNA products were measured by RT-PCR. A minimum number of three measurements were used to calculate each mean. Graph shows percent exon inclusion as a function of the KCl treatment and differentiation of the cultures. Representative results are shown in the gel panels. The percent change in exon inclusion (ΔEI) was calculated as follows: the percent exon inclusion in the 50 mM KCl sample minus the percent exon inclusion in the 0 mM KCl sample.</p

Biochemical Analysis of Protein Binding to UAGG Silencer Motifs as a Function of Cell Excitation

<div><p>(A) Schematic of methodology used to analyze protein binding to RNA substrates in nuclear extracts from KCl-induced and mock-treated cultures. UV crosslinking assays were performed under splicing conditions to monitor protein binding to radiolabeled RNA oligos (lanes 1–8), and to the full-length CI cassette exon (lanes 9 and 10). Sequences of RNA oligos and schematic of CI exon are shown at right of gel panels. Gel panels show radiolabeled proteins after SDS-PAGE separation; kDa ladder indicates molecular weight standards.</p> <p>(B) Affinity selection of proteins non-covalently bound to RNA substrates containing the full-length CI cassette exon with (M3_E18) or without (E18) the M3 hairpin. Assembly reactions containing RNA substrates pre-bound to MS2-MBP were incubated under splicing conditions, bound to amylose columns, and eluted with maltose. Samples were resolved by SDS-PAGE and transferred to nylon membranes for immunoblotting with hnRNP A1-specific 9H10 (lanes 11–16) or ASF/SF2 (lanes 17–22) antibody. A1 and A1^B represent the major and minor isoforms of hnRNP A1, respectively.</p> <p>(C) Quantitative Western blots show serial dilutions of recombinant hnRNP A1 (MBP-A1) grown in Escherichia coli as reference standards from 1 to 20 ng (top panel). Nuclear levels of hnRNP A1 and A1B are shown at three loading levels, 100, 200, and 400 ng of total nuclear extract (NE) protein (bottom panel). Nuclear extracts were prepared from mock-treated (Mock) and 50 mM KCl-treated cultures (Depol). Monoclonal antibody 9H10 was used to detect hnRNP A1 in both blots.</p></div

Summary of Exon-Skipping Responsiveness and UAGG Code

<div><p>(A) Percent exon inclusion values are shown graphically for the samples represented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-g008" target="_blank">Figure 8</a>.</p> <p>(B) Percent ΔEI values represent exon-skipping responses to depolarization for samples in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050036#pbio-0050036-g008" target="_blank">Figure 8</a>. Negative values (≤−10%) reflect significant exon skipping, whereas positive values (≥10%) reflect exon inclusion. The number of UAGG (hnRNP A1-type) silencing motifs are indicated (bottom).</p></div

Promoters from Transgenic Mice Drive Neuron- and Glial-Specific Expression in Cortical Cultures

<div><p>(A) Schematic of reporter constructs and results of fluorescent reporter overlap with molecular markers. Full-length (8.5 kb; CaMKII_279), and shortened (2.2 kb; CaMKII_22) mouse alpha CaMKII promoters were fused to the open reading frame of EYFP (constructs 1 and 2). Restriction sites used for the promoter deletions or for insertion of the fluorescent reporter coding sequence are indicated. The glial-specific Gfa2 promoter was fused to DsRed (construct 3). The CMV promoter fused to EYFP (construct 4) or DsRed (construct 5) were used as controls. Numbers at right indicate the percentage of fluorescent cells that showed overlap expression with NeuN or GFAP antibody staining. For each percentage, the total number of fluorescent cells counted for antibody staining <i>(n)</i> is indicated.</p> <p>(B) Summary of experimental scheme and representative results. Following transfection, cortical cultures were fixed and stained with antibody for molecular marker as indicated using a FITC or TRITC conjugated secondary antibody (left panel). To measure overlap expression, cells expressing the EYFP (or DsRed) fluorescent reporter were localized, then switched to the channel for detection of TRITC (or FITC) label. Representative results are shown for CaMKII and Gfa2 reporters (right panels). Cells expressing CaMKII EYFP reporters are green; antibody staining is red (TRITC-anti-Neu N, top panels; TRITC-anti-GFAP, bottom panels). Note that the NeuN marker is restricted to the cell nucleus as expected. Cells expressing the Gfa2 DsRed reporter are red; antibody staining is green. Yellow signifies expression overlap.</p> <p>(C) Graphical summary of the fluorescent reporter expression. The <i>y</i>-axis represents the percent expression overlap of the fluorescent reporter and antibody staining for neurons (NeuN) or glia (GFAP). The <i>x</i>-axis represents promoter constructs from (A).</p></div

Transfer of Multicomponent UAGG Silencing Motif Pattern Confers Sensitivity of a Constitutive Exon to Depolarization in Primary Neurons

<div><p>(A) The schematic at top illustrates the heterologous splicing reporters used for the transfer experiments. Exon 5 of the DIP13 transcript and 12 nucleotides of each flanking intron was inserted between the NdeI and XbaI restriction sites of the SIRT1a_CaMKII22 plasmid. An engineered EcoR1 site is indicated by the arrowhead in the sequences shown. Intron/exon lengths (nucleotides) are as follows: exon 1, 308; intron 1, 340; exon 2, 94; intron 2, 287; and exon 3, 436. For the middle exon sequences shown in the expanded region for wild-type exon 5, the predicted exonic enhancer motifs are highlighted in black letters. UAGG silencing motifs were introduced into the middle exon of the DIP_3aG, and DIP_E2 splicing reporters as indicated (red letters); mutations are underscored. Colon indicates 5′ splice site cleavage position. The first 12 nucleotides of the downstream intron are shown at right for the wild-type substrate (DIP13_E5). The GGGG motif at intron nucleotides 6–9 is highlighted in red.</p> <p>(B) Representative results of RT-PCR analysis and values for percent exon inclusion and <b>Δ</b>EI are shown; the exon-included (<<) and -skipped (<) mRNAs are indicated. SD, standard deviation.</p></div

Context-adaptive based CU processing for 3D-HEVC

<div><p>The 3D High Efficiency Video Coding (3D-HEVC) standard aims to code 3D videos that usually contain multi-view texture videos and its corresponding depth information. It inherits the same quadtree prediction structure of HEVC to code both texture videos and depth maps. Each coding unit (CU) allows recursively splitting into four equal sub-CUs. At each CU depth level, it enables 10 types of inter modes and 35 types of intra modes in inter frames. Furthermore, the inter-view prediction tools are applied to each view in the test model of 3D-HEVC (HTM), which uses variable size disparity-compensated prediction to exploit inter-view correlation within neighbor views. It also exploits redundancies between a texture video and its associated depth using inter-component coding tools. These achieve the highest coding efficiency to code 3D videos but require a very high computational complexity. In this paper, we propose a context-adaptive based fast CU processing algorithm to jointly optimize the most complex components of HTM including CU depth level decision, mode decision, motion estimation (ME) and disparity estimation (DE) processes. It is based on the hypothesis that the optimal CU depth level, prediction mode and motion vector of a CU are correlated with those from spatiotemporal, inter-view and inter-component neighboring CUs. We analyze the video content based on coding information from neighboring CUs and early predict each CU into one of five categories i.e., DE-omitted CU, ME-DE-omitted CU, SPLIT CU, Non-SPLIT CU and normal CU, and then each type of CU adaptively adopts different processing strategies. Experimental results show that the proposed algorithm saves 70% encoder runtime on average with only a 0.1% BD-rate increase on coded views and 0.8% BD-rate increase on synthesized views. Our algorithm outperforms the state-of-the-art algorithms in terms of coding time saving or with better RD performance.</p></div